Showing papers by "Rockefeller University published in 1989"

Hematopoietic reconstitution in a patient with fanconi's anemia by means of umbilical-cord blood from an hla-identical sibling

Abstract: The clinical manifestations of Fanconi’s anemia, an autosomal recessive disorder, include progressive pancytopenia, a predisposition to neoplasia, and nonhematopoietic developmental anomalies [1-3]. Hypersensitivity to the clastogenic effect of DNA-cross-linking agents such as diepoxybutane acts as a diagnostic indicator of the genotype of Fanconi’s anemia, both prenatally and postnatally [3-6]. Prenatal HLA typing has made it possible to ascertain whether a fetus is HLA-identical to an affected sibling [7]. We report here on hematopoietic reconstitution in a boy with severe Fanconi’s anemia who received cryo-preserved umbilical-cord blood from a sister shown by prenatal testing to be unaffected by the disorder, to have a normal karyotype, and to be HLA-identical to the patient. We used a pretransplantation conditioning procedure developed specifically for the treatment of such patients [8]; this technique makes use of the hypersensitivity of the abnormal cells to alkylating agents that cross-link DNA [9,10] and to irradiation [11] In this case, the availability of cord blood obviated the need for obtaining bone marrow from the infant sibling. This use of cord blood followed the suggestion of one of us that blood retrieved from umbilical cord at delivery, usually discarded, might restore hematopoiesis – a proposal supported by preparatory studies by some of us [12] and consistent with reports on the presence of hematopoietic stem and multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in human umbilical-cord blood (see the references cited by Broxmeyer et al. [12]).

Columnar specificity of intrinsic horizontal and corticocortical connections in cat visual cortex

Abstract: A prominent and stereotypical feature of cortical circuitry in the striate cortex is a plexus of long-range horizontal connections, running for 6-8 mm parallel to the cortical surface, which has a clustered distribution. This is seen for both intrinsic cortical connections within a particular cortical area and the convergent and divergent connections running between area 17 and other cortical areas. To determine if these connections are related to the columnar functional architecture of cortex, we combined labeling of the horizontal connections by retrograde transport of rhodamine-filled latex microspheres (beads) and labeling of the orientation columns by 2-deoxyglucose autoradiography. We first mapped the distribution of orientation columns in a small region of area 17 or 18, then made a small injection of beads into the center of an orientation column of defined specificity, and after allowing for retrograde transport, labeled vertical orientation columns with the 2-deoxyglucose technique. The retrogradely labeled cells were confined to regions of orientation specificity similar to that of the injection site, indicating that the horizontal connections run between columns of similar orientation specificity. This relationship was demonstrated for both the intrinsic horizontal and corticocortical connections. The extent of the horizontal connections, which allows single cells to integrate information over larger parts of the visual field than that covered by their receptive fields, and the functional specificity of the connections, suggests possible roles for these connections in visual processing.

Origin of luteinizing hormone-releasing hormone neurons.

Abstract: Neurons expressing luteinizing hormone-releasing hormone (LHRH), found in the septal-preoptic nuclei and hypothalamus, control the release of gonadotropic hormones from the anterior pituitary gland and facilitate reproductive behaviour. LHRH-expressing neurons are also found in the nervus terminalis, a cranial nerve that is a part of the accessory olfactory system and which projects directly from the nose to the septal-preoptic nuclei in the brain. During development, LHRH-immunoreactivity is detected in the peripheral parts of the nervus terminalis before it is found in the brain. Using a combination of LHRH immunocytochemistry and tritiated thymidine autoradiography in fetal mice, we show that LHRH neurons originate in the medial olfactory placode of the developing nose, migrate across the nasal septum and enter the forebrain with the nervus terminalis, arching into the septal-preoptic area and hypothalamus. Clinically, this migratory route for LHRH-expressing neurons could explain the deficiency of gonadotropins seen in 'Kallmann's syndrome' (hypogonadotropic hypogonadism with anosmia).

Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes.

Abstract: Psoriasis is a common papulosquamous skin disease. The histopathology is characterized by epidermal hyperplasia and inflammation. Recent studies suggest that keratinocyte proliferation and inflammation in psoriasis are manifestations of the same underlying pathological process. Interleukin 6 (IL-6), a cytokine that is a major mediator of the host response to tissue injury and infection, is produced by both keratinocytes and leukocytes in culture. IL-6 expression was studied in psoriatic plaques by immunoperoxidase staining with two different polyclonal anti-recombinant IL-6 antisera and by in situ nucleic acid hybridization with IL-6 cRNA probes. Epidermal and dermal cells in active psoriatic plaques from 35 psoriasis patients stained heavily for IL-6 as compared with nonlesional skin and with plaques after treatment with antimetabolic and antiinflammatory agents. Absorption of the anti-recombinant IL-6 antisera with purified fibroblast-derived IL-6 or with recombinant IL-6, but not bovine serum albumin, removed the immunostaining. Increased levels of IL-6 were detected in the plasma of patients with active psoriasis (mean 3 ng/ml) by using two different bioassays. IL-6 production by proliferating keratinocytes was suggested by IL-6-specific immunostaining in cultured normal and psoriatic keratinocytes and by the detection of mRNA specific for IL-6 in psoriatic epidermis by in situ hybridization. IL-6 stimulated the proliferation of cultured, normal human keratinocytes as assessed by two different assays. Thus, IL-6 could directly contribute to the epidermal hyperplasia seen in psoriatic epithelium as well as affect the function of dermal inflammatory cells.

Streptococcal M protein: molecular design and biological behavior.

Abstract: M protein is a major virulence determinant for the group A streptococcus by virtue of its ability to allow the organism to resist phagocytosis. Common in eucaryotes, the fibrillar coiled-coil design for the M molecule may prove to be a common motif for surface proteins in gram-positive organisms. This type of structure offers the organism several distinct advantages, ranging from antigenic variation to multiple functional domains. The close resemblance of this molecular design to that of certain mammalian proteins could help explain on a molecular level the formation of epitopes responsible for serological cross-reactions between microbial and mammalian proteins. Many of the approaches described in the elucidation of the M-protein structure may be applied for characterizing similar molecules in other microbial systems. Images

Common amino acid sequence domains among the LEA proteins of higher plants.

Abstract: LEA proteins are late embryogenesis abundant in the seeds of many higher plants and are probably universal in occurrence in plant seeds. LEA mRNAs and proteins can be induced to appear at other stages in the plant's life by desiccation stress and/or treatment with the plant hormone abscisic acid (ABA). A role in protecting plant structures during water loss is likely for these proteins, with ABA functioning in the stress transduction process. Presented here are conserved tracts of amino acid sequence among LEA proteins from several species that may represent domains functionally important in desiccation protection. Curiously, an 11 amino acid sequence motif is found tandemly repeated in a group of LEA proteins of vastly different sizes. Analysis of this motif suggests that it exists as an amphiphilic α helix which may serve as the basis for higher order structure.

Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis.

Abstract: Endothelial cells play a critical role in thromboregulation by virtue of a surface-connected fibrinolytic system. Cultured endothelial cells synthesize and secrete tissue-type plasminogen activator (t-PA) which can bind to at least two discrete sites on the cell surface. These binding sites preserve the catalytic activity of t-PA and protect it from its physiological inhibitor (PAI-1). N-terminal glutamic acid plasminogen (Glu-PLG), the main circulating fibrinolytic zymogen, also interacts specifically with the endothelial cell surface. Binding is associated with a 12-fold increase in catalytic efficiency of plasmin generation by t-PA which may reflect conversion of Glu-PLG to its plasmin-modified form, N-terminal lysine plasminogen (Lys-PLG). Lipoprotein(a) is an atherogenic lipoprotein particle which contains the plasminogen-like apolipoprotein(a) bound to low density lipoprotein. We report here that lipoprotein(a) interferes with endothelial cell fibrinolysis by inhibiting plasminogen binding and hence plasmin generation. In addition, we demonstrate lipoprotein(a) accumulation in atherosclerotic lesions. These findings may provide a link between impaired cell surface fibrinolysis and progressive atherosclerosis.

Influences of hippocampal place cell firing in the awake state on the activity of these cells during subsequent sleep episodes

Abstract: Rat hippocampal (CA1) complex spike “place cells” of freely behaving rats were recorded in pairs continuously during a series of waking (exploration and still-alert), drowsy (quiet-awake), and sleeping (slow- wave, pre-rapid-eye-movement and rapid-eye-movement sleep) behaviors. Pairs of units were selected that had nonoverlapping place fields. The rats were restricted from entering the place field of either cell overnight, and on the day of recording cells were exposed to their individual place fields independently and in a counterbalanced manner. Following exposure, recordings were made in the subsequent sleep episodes and the firing characteristics of both cells were analyzed. Following exposure, significant increases in the spiking activity of the exposed cell were observed in the subsequent sleeping states that were not evident in the unexposed cell. The increased activity was observed in the rate of firing (spikes/sec), the rate of occurrence of bursts with multiple spikes, as well as the number of bursts displaying short (2–4 msec) interspike intervals. The findings suggest that neuronal activity of hippocampal place cells in the awake states may influence the firing characteristics of these cells in subsequent sleep episodes. The increased firing rates along with the greater number of multiple spike bursts and the shorter interspike intervals within the burst, following exposure to a cell's place field, may represent possible information processing during sleep.

Presentation of exogenous protein antigens by dendritic cells to T cell clones. Intact protein is presented best by immature, epidermal Langerhans cells.

Abstract: The capacity of dendritic cells to present protein antigens has been studied with two MHC class II-restricted, myoglobin-specific, T cell clones. Spleen dendritic cells and cultured epidermal Langerhans cells (LC) presented native myoglobin weakly and often not at all. These same populations were powerful stimulators of allogeneic T cells in the primary MLR. Freshly isolated LC were in contrast very active in presenting proteins to T cell clones but were weak stimulators of the MLR. Both fresh and cultured LC could present specific peptide fragments of myoglobin to the clones. These results suggest that dendritic cells in nonlymphoid tissues like skin can act as sentinels for presenting antigens in situ, their accessory function developing in two phases. First antigens are captured and appropriately presented. Further handling of antigen then is downregulated while the cells acquire strong sensitizing activity for the growth and function of resting T lymphocytes. The potent MLR stimulating activity of cultured epidermal LC and lymphoid dendritic cells probably reflects prior handling of antigens leading to the formation of allogeneic MHC-peptide complexes.

The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns.

Abstract: We have analyzed expression conferred by two domains from the cauliflower mosaic virus (CaMV) 35S promoter and found different patterns in seeds, seedlings and seven week old plants. Expression from domain A (-90 to +8) is strongest in the radicle of the embryo, the radicle pole of the endosperm and in root tissue of seedlings and mature plants. Expression from domain B (-343 to -90) is strongest in the cells adjacent the cotyledon of the endosperm, in the cotyledons of the embryo and seedings and in the leaves and stem of mature plants. When both domain A and domain B are present expression is detectable in most tissues at all stages of development. Thus analysis of a constitutive promoter in transgenic plants can be used to identify cis elements that confer tissue specific and developmentally regulated expression.

Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes.

Abstract: Transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) are expressed at high levels in the liver and also in at least one other cell type. We report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the alpha 1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. We had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and alpha 1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the result of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and alpha 1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

Synapsins: Mosaics of Shared and Individual Domains in a Family of Synaptic Vesicle Phosphoproteins

Abstract: Synapsins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. Molecular cloning reveals that the synapsins comprise a family of four homologous proteins whose messenger RNA's are generated by differential splicing of transcripts from two genes. Each synapsin is a mosaic composed of homologous amino-terminal domains common to all synapsins and different combinations of distinct carboxyl-terminal domains. Immunocytochemical studies demonstrate that all four synapsins are widely distributed in nerve terminals, but that their relative amounts vary among different kinds of synapses. The structural diversity and differential distribution of the four synapsins suggest common and different roles of each in the integration of distinct signal transduction pathways that modulate neurotransmitter release in various types of neurons.

Macrophage inflammatory proteins 1 and 2: members of a novel superfamily of cytokines.

Abstract: A number of studies of inflammation and of cell growth and transformation have recently converged by defining two related families of cytokines. The first, represented by macrophage inflammatory protein 1, is composed of several gene products that have been identified in activated T cells, macrophages, and fibroblasts. The biological activities of this family are still being characterized but so far include effects on neutrophils, monocytes, and hematopoietic cells. The second, represented by macrophage inflammatory protein 2, includes platelet products such as platelet factor 4 and beta-thromboglobulin as well as several other recently described gene products that have effects on a number of cell types including neutrophils, fibroblasts, hematopoietic cells, and melanoma cells. The two families are structurally related and may have evolved from a common ancestral gene that duplicated and then diverged. Their differential control and expression in a wide variety of cell types suggests that they may have m...

Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants

Abstract: The 35S promoter of cauliflower mosaic virus (CaMV) is able to confer high-level gene expression in most organs of transgenic plants. A cellular factor from pea and tobacco leaf tissue, which recognizes nucleotides in a tandemly repeated TGACG motif at the -75 region of this promoter, has been detected by DNase I footprinting and gel retardation assays. This factor is named activation sequence factor 1 (ASF-1). A cellular factor binding to the two TGACG motifs can also be detected in tobacco root extracts. Mutations at these motifs inhibit binding of ASF-1 to the 35S promoter in vitro. When examined in transgenic tobacco, these mutations cause a 50% drop in leaf expression of the 35S promoter. In addition, these same mutations attenuate stem and root expression of the 35S promoter about 5- to 10-fold when compared to the level of expression in leaf. In contrast, mutations at two adjacent CCAAT-box-like sequences have no dramatic effect on promoter activity in vivo. A 21-base-pair element containing the two TGACG motifs is sufficient for binding of ASF-1 in vitro when inserted in a green-tissue-specific promoter. In vivo, the insertion of an ASF-1 binding site caused high levels of expression in root. Thus, a single factor binding site that is defined by site-specific mutations is shown to be sufficient to alter the expression pattern of promoters in vivo.

Identification and characterization of macrophage inflammatory protein 2

Abstract: In response to endotoxin, macrophages secrete a protein with a molecular mass of approximately 6000 Da and with an affinity for heparin. This protein, which we term "macrophage inflammatory protein 2," is a potent chemotactic agent for human polymorphonuclear leukocytes. In addition, subcutaneous administration of the monokine causes a localized inflammatory reaction. Partial N-terminal sequence data reveal similarity to a family of proteins, the archetype of which is platelet factor 4. Although macrophage inflammatory protein 2 is a distinct member of the platelet factor 4 family, its sequence is most closely related to that of the gro/KC gene product, which is expressed in transformed or platelet-derived growth factor-treated cells.

Two tobacco DNA-binding proteins with homology to the nuclear factor CREB

Abstract: THE 35S promoter of the cauliflower mosaic virus (CaMV) contains a tandem repeat of the sequence TGACG in the region −83 to −63. This 21-base pair (bp) sequence, called as-1, is involved in root expression of the 35S promoter. When inserted in a promoter of a gene expressed specifically in photosynthetic tissues, as-1 confers high level expression in roots13. We have described a factor, ASF-1, that binds specifically to as-1 in vitro. There is a good correlation between ASF-1 binding affinity to as-1 related sequences in vitro and the function of these sequences in vivo. These results strongly suggest that ASF-1 is responsible for the function of as-1 (ref. 13). Here we report the isolation of tobacco complementary DNA clones encoding two TGACG-sequence-specific binding-proteins (TGAla and TGAlb). Sequence analysis of the cDNA clones shows that both proteins contain a basic region that shows high homology to a stretch of basic amino acids in the nuclear factors CREB, GCN4, and c-Jun1-4 to a 'leucine-zipper' region5. On the basis of binding specificity we propose TGAla to be a good candidate for ASF-1.

Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins.

Abstract: The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapic, ferulic and caffeic acids) are cinnamic acid derivatives that have several pratical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These new matrices are produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).

Long-term potentiation in the motor cortex.

Abstract: Long-term potentiation (LTP) is a model for learning and memory processes Tetanic stimulation of the sensory cortex produces LTP in motor cortical neurons, whereas tetanization of the ventrolateral nucleus of the thalamus, which also projects to the motor cortex, does not However, after simultaneous high-frequency stimulation of both the sensory cortex and the ventrolateral nucleus of the thalamus, LTP of thalamic input to motor cortical neurons is induced This associative LTP occurs only in neurons in the superficial layers of the motor cortex that receive monosynaptic input from both the sensory cortex and the ventrolateral nucleus of the thalamus Associative LTP in the motor cortex may constitute a basis for the retention of motor skills

Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages.

Abstract: Lipopolysaccharide binding protein (LBP) is an acute-phase reactant that binds bacterial LPS. We show that LBP binds to the surface of live Salmonella and to LPS coated erythrocytes (ELPS), and strongly enhances the attachment of these particles to macrophages. LBP bridges LPS-coated particles to macrophages (MO) by first binding to the LPS, then binding to MO. Pretreatment of ELPS with LBP enabled binding to MO, but pretreatment of MO had no effect. Moreover, MO did not recognize erythrocytes coated with LBP unless LPS was also added, thus suggesting that interaction of LBP with LPS results in a conformational change in LBP that allows recognition by MO. Binding of LBP-coated particles appears to be mediated by a receptor found on blood monocytes and MO but not on other leukocytes or umbilical vein endothelium. The receptor is mobile in the plane of the membrane since binding activity on MO was downmodulated upon spreading of cells on surfaces coated with LBP-LPS complexes. The receptor appears to be distinct from other opsonic receptors since downmodulation of CR1, CR3, Fc gamma RI, Fc gamma RII, and Fc gamma RIII with mAbs did not affect binding of LBP-coated particles, and leukocytes from CD18-deficient patients bound LBP-coated particles normally. Coating of erythrocytes with LBP-LPS complexes strongly enhanced phagocytosis observed in the presence of suboptimal amounts of anti-erythrocyte IgG. However, binding mediated by LBP-LPS complexes alone caused neither phagocytosis of the LBP-coated erythrocytes nor initiation of an oxidative burst. The results of our studies define LBP as an opsonin. During the acute phase, LBP can be expected to bind gram-negative bacteria and bacterial fragments and promote the interaction of coated bacteria with phagocytes.

A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions

Abstract: We have raised an mAb to a previously undescribed 135-kD externally disposed integral membrane protein that is enriched in the intercellular junctional domain of cultured human umbilical vein endothelial cells. This protein localizes at the appositional surfaces of cells as they become confluent and is stably expressed in the junctional zones of confluent monolayers. This protein is expressed in situ on continuous endothelia of all blood vessels in all human tissues examined. Moreover, this protein, as determined by mAb immunocytochemistry, is not expressed by any other cell type. This protein may mediate endothelial-specific functions restricted to the intercellular domain. It may also serve as a unique cell surface marker for the identification and purification of human endothelial cells.

Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants.

Abstract: The 35S promoter is a major promoter of the cauliflower mosaic virus that infects crucifers. This promoter is still active when excised from cauliflower mosaic virus and integrated into the nuclear genome of transgenic tobacco. Previous work has shown that the -343 to -46 upstream fragment is responsible for the majority of the 35S promoter strength (Odell, J.T., Nagy, F., and Chua, N.-H. [1985]. Nature 313, 810-812). Here we show by 5', 3', and internal deletions that this upstream fragment can be subdivided into three functional regions, -343 to -208, -208 to -90, and -90 to -46. The first two regions can potentiate transcriptional activity when tested with the appropriate 35S promoter sequence. In contrast, the -90 to -46 region by itself has little activity but it plays an accessory role by increasing transcriptional activity of the two distal regions. Finally, we show that monomers and multimers of a 35S fragment (-209 to -46) can act as enhancers to potentiate transcription from a heterologous promoter.

Regulated genes in transgenic plants.

Abstract: Transgenic plants are an effective system for the study of regulated gene expression. Developmental control of expression can be monitored by assaying different tissues or by assaying a plant at different developmental stages. Analysis of the petunia 5-enolpyruvylshikimate-3-phosphate synthase gene, which is highly expressed in flowers, allowed identification of an upstream region that confers tissue-specific and developmentally regulated expression. The cell specificity of expression in floral tissues has been defined by histochemical localization. This expression is contrasted to that of the 35S promoter of cauliflower mosaic virus, a nominally constitutive promoter that shows a definite specificity of expression in floral tissues. Moreover, this expression differs in transgenic hosts of different species.

Reduction of inflammation, tissue damage, and mortality in bacterial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leukocytes.

Abstract: We tested if specific inhibition of recruitment of leukocytes across the blood brain barrier from the vascular compartment to the cerebrospinal fluid (CSF) space reduced tissue damage and improved the outcome of infection in a rabbit model of experimental meningitis. The CD11/CD18 complex of receptors on leukocytes promotes adhesion of these cells to endothelia, a process required for egress of cells into the extravascular space. Intravenous injection of the anti-CD18 mAb IB4 effectively blocked the development of leukocytosis in the CSF of animals challenged intracisternally with living bacteria, bacterial endotoxin, or bacterial cell wall. This effect was associated with protection from blood brain barrier injury as measured by exclusion of serum proteins from CSF in mAb-treated animals. The densities of bacteria in CSF and the degrees of bacterial killing due to ampicillin were not affected by the antibody. Animals receiving the antibody experienced a delay in the development of bacteremia and a significantly reduced inflammatory response during ampicillin-induced bacterial killing. Therapy with mAb IB4 prevented development of brain edema and death in animals challenged with lethal doses of Streptococcus pneumoniae. These studies indicate that the major mechanism of leukocyte migration across the blood brain barrier involves the CD11/CD18 receptors and that inflammatory leukocytes recruited by this mechanism are a major cause of blood brain barrier injury and cerebral edema during meningitis.

Factors affecting the ultraviolet laser desorption of proteins

Abstract: The production of high-mass quasimolecular ions from proteins by matrix-assisted ultraviolet laser desorption is described. A simple time-of-flight system using a Q-switched frequency-quadrupled Nd-YAG laser to desorb protein molecules is shown to have a mass range of up to 116,000 u by the observation of intact, singly charged quasimolecular ions from 700 fmol of beta-galactosidase subunit (mol.wt = 116,336 Da). Both positive- and negative-ion spectra of proteins are shown. Four new matrix materials, with properties as good as or better than nicotinic acid, are described. A mass resolution of approximately 500 (full width at half maximum definition) is demonstrated for proteins with mol.wt less than 20,000 Da. Product species, formed by fast photochemical reactions in the matrix, are observed to form adduct ions with protein molecules. These adduct ions are a significant cause of the observed broadness of protein quasimolecular ion peaks. The practical physical considerations in detection of large-mass quasimolecular ions from laser desorption, such as detector overloading, are discussed.

HIV-1 Infection Among Intravenous Drug Users in Manhattan, New York City, From 1977 Through 1987

Abstract: Intravenous drug users are the second largest group to develop the acquired immunodeficiency syndrome, and they are the primary source for heterosexual and perinatal transmission in the United States and Europe. Understanding long-term trends in the spread of human immunodeficiency virus among intravenous drug users is critical to controlling the acquired immunodeficiency syndrome epidemic. Acquired immunodeficiency syndrome surveillance data and seroprevalence studies of drug treatment program entrants are used to trace seroprevalence trends among intravenous drug users in the borough of Manhattan. The virus entered this drug-using group during the mid-1970s and spread rapidly in 1979 through 1983. From 1984 through 1987, the seroprevalence rate stabilized between 55% and 60%—well below hepatitis B seroprevalence rates. This relatively constant rate is attributed to new infections, new seronegative persons beginning drug injection, seropositive persons leaving drug injection, and increasing conscious risk reduction. ( JAMA 1989;261:1008-1012)

New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity.

Abstract: Seventeen clinical isolates of Staphylococcus aureus (from the United States and Europe) selected for low (borderline)-level methicillin resistance (MIC of methicillin, 2 to 4 micrograms/ml; MIC of oxacillin, 0.5 to 8 micrograms/ml) were examined for their mechanisms of resistance. Five strains were typical of heterogeneous S. aureus: they gave positive reactions with a DNA probe specific for mec and contained a small fraction (10(-6] of highly resistant cells (MIC, greater than 100 micrograms/ml). The rest of the 12 strains were homogeneous with respect to their methicillin resistance: the MIC of methicillin for all cells was 2 to 4 micrograms/ml, and no cells for which MICs were 50 micrograms/ml or higher were detectable (less than 10(-9]. None of these strains reacted with the mec-specific DNA probe. One representative strain of each group was characterized in more detail. Strain CDC-1, prototype of heterogeneous methicillin-resistant S. aureus, contained penicillin-binding protein (PBP) 2a; its DNA could transform a methicillin-susceptible and novobiocin-resistant recipient to methicillin resistance with ca. 35% linkage to Novr. Introduction of the "factor X" determinant (K. Murakami and A. Tomasz, J. Bacteriol. 171:874-879, 1989) converted strain CDC-1 to high, homogeneous resistance. Strain CDC-6, prototype of the second group of isolates, showed completely homogeneous MICs of methicillin, oxacillin, and cefotaxime. The strain contained modified "normal" PBPs: PBPs 1 and 2 showed low drug reactivity (and/or cellular amounts), and PBP 4 was present in elevated amounts. No PBP 2a could be detected. DNA isolated from strain CDC-6 could transform the methicillin-susceptible and novobiocin-resistant strain to methicillin resistance in a multistep fashion, but this resistance showed no genetic linkage to the Nov marker. We suggest that staphylococci with borderline resistance may contain at least three different classes of mechanism: heterogeneous, methicillin-resistant S. aureus, PBPs of modified drug reactivities, and the previously reported hyperproduction of beta-lactamase (L.K. McDougal and C. Thornsberry, J. Clin Microbiol. 23:832-839, 1986). Images