Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.

Increase in intracellular Na+: transmembrane signal for rejoining of DNA strand breaks in proliferating lymphocytes.

Two early events in the mitogen-induced entry of murine splenocytes into proliferation are (a) a rapid rise in influx of Na+, causing its total internal concentration to increase by 42 +/- 7% within 2 h of culture with concanavalin A (Con A), and (b) rejoining of some 3000 DNA strand breaks per diploid genome within the same period. Con A did not induce rejoining in low Na+ (less than 9 mM) medium, but the process began directly when Na+ was added, at its usual concentration, to the growth medium. Incubation of cells with ouabain, an inhibitor of the Na+K+-ATPase, or monensin, a Na+ ionophore, caused an increase in the internal Na+ concentration in normal, but not in low, Na+ medium. In the former (but not in the latter) medium, both ouabain and monensin caused rejoining of the DNA strand breaks to occur in resting lymphocytes, i.e., in the absence of mitogen. Stimulation of splenocytes with Con A also resulted in a rapid but transient increase in the level of intracellular free Ca2+. This effect was also observed in the absence of extracellular Na+; however, deprivation of extracellular Ca2+ completely abolished this effect. Moreover, the intracellular free Ca2+ level was significantly higher in cells suspended in Na+-free buffer or medium. Since the Con A-induced rejoining of DNA strand breaks occurred in the absence of extracellular Ca2+ and removal of extracellular Na+ had no inhibitory effect on the Con A-induced increase in the level of intracellular free Ca2+, the Con A-stimulated repair could not have been mediated by the initial increase in Ca2+ influx. The early mitogen-induced increase in the internal Na+ concentration is a necessary and sufficient signal for the rejoining of breaks, an event that must occur before the proliferating lymphocytes can replicate their DNA.

The beta-glucosidase of the yeast cell surface.

There are two distinct components of the system which limits the rate at which intact cells of S. cerevisiae C hydrolyze external beta-glucosides; one component requires metabolic energy and the other is stereospecific for beta-glucosides. The stereospecific component is localized at the cell membrane, as shown by its sensitivity to heavy metal inhibitors which did not penetrate the cell under the conditions used. It was shown that cellobiose-grown cells were able to remove cellobiose from the medium in which they were incubated, and that the cellobiose uptake system was identical to that which limits the patent beta-glucosidase activity. In order to test the hypothesis that the system in question was a transport system, for beta-glucosides the ability of cellobiose-grown cells to take up (14)C-labeled methyl-beta-glucoside (MBG) was studied. The induced cells were able to take up MBG-(14)C and the label could be partially chased out by cold MBG and cellobiose; glucose-grown cells could not incorporate label. However, induced cells could not take up label when incubated with (14)C-MBG, thus excluding the hypothesis of transport of intact beta-glucosides. It was concluded that the stereospecific membrane component was actually a beta-glucosidase, coupled to an energy-dependent transport system for the glucose moiety; the function of the latter was rate-limiting in the over-all activity of the entire system.

Hereditary neuropathy with upper motor-neuron, visual pathway, and autonomic disorders.

A 42-year-old man had progressive distal weakness and muscle atrophy, stocking-type sensory loss, upper motor-neuron and visual pathway lesions, and dysautonomia. Electrodiagnostic tests revealed a generalized sensorimotor peripheral neuropathy that largely involved axons. Low recumbent and upright norepinephrine levels implied a peripheral autonomic defect. Sural nerve biopsy showed mild abnormalities of medium and small size fibers. The patient's mother and two brothers were also affected. Other causes of peripheral motor, sensory, and autonomic failure were eliminated. This kinship does not fit any generally accepted classification of hereditary neuropathies.

Infectious agents in spinal epidural abscesses.

Of 27 cases of spinal epidural abscess, 19 were caused by bacteria, 7 by Mycobacterium tuberculosis, and 1 by Echinococcus granulosus. Blunt trauma and cutaneous infections were the most frequent preceding events in bacterial cases. Tuberculous abscess was usually the sole manifestation of reactivation of dormant tuberculosis. Drug addiction, the most common cause in young adults, was associated with gram-negative infections. Whatever the infectious agent, paraparesis for longer than 4 days led to a poor outcome. Myelography was the best diagnostic test, whereas lumbar puncture and percutaneous bone biopsy offered little specific information.

Relapsing ophthalmoparesis-sensory neuropathy syndrome.

We studied two patients with recurrent sensory neuropathy and weakness of extraocular muscles. There was electrophysiologic evidence of multifocal demyelination of sensory nerves, with relative sparing of somatic motor nerves. Sural nerve biopsy in one patient showed segmental demyelination. We believe that these patients had an unusual form of inflammatory polyneuropathy--possibly a relapsing variant of the Miller-Fisher syndrome of acute idiopathic polyneuritis.

Sensory neuropathy associated with Dursban (chlorpyrifos) exposure.

Chlorpyrifos (Dursban) is an organophosphate insecticide with extensive domestic and agricultural applications. It is regarded as safe for these purposes; one report of neurotoxicity is attributed to massive ingestion in a suicide attempt. We report eight people who developed peripheral neuropathy after exposure to exterminator-applied commercial Dursban; five also experienced memory loss and cognitive slowing. Evaluation failed to reveal other causes of neurologic dysfunction; symptoms recurred in one patient following accidental reexposure. We conclude that environmental contact with chlorpyrifos can cause sensory neuropathy and CNS dysfunction and that this agent should be used with caution.

Distal axonopathy associated with chronic gluten enteropathy: a treatable disorder.

Our experience with one patient and literature review reveals that patients with chronic gluten enteropathy and severe steatorrhea may develop a generalized peripheral neuropathy of the distal axonopathy type. Vitamin levels are usually normal, and the neuropathy appears to respond to gluten restriction.

Spinal epidural lipomatosis: a serious complication of iatrogenic Cushing's syndrome.

Our experience and review of the literature demonstrate that spinal epidural lipomatosis is a rare but serious neurologic complication of iatrogenic Cushing's syndrome. Most patients develop slowly progressive paraparesis over months, but a subgroup exists which presents with acute, irreversible paraplegia. We consider therapeutic options.

Luetic meningitis with gumma: clinical, radiographic, and neuropathologic features.

A 28-year-old man had headache and unilateral third-nerve palsy as the presenting features of acute syphilitic meningitis, accompanied by an asymptomatic cerebral gamma.

Progressive dementia, visual deficits, amyotrophy, and microinfarcts.

Data from three patients and 22 previously reported cases suggest that cerebral microinfarction causes a recognizable clinical syndrome. All cases present with stroke, followed by progressive dementia and often with visual field deficits, peripheral vascular disease, and signs of motor neuron dysfunction. The average age at onset is 45, and most patients have been men. Many patients have had valvular or ischemic heart disease; in one of our cases, mitral stenosis caused embolic microinfarcts.

Studies of the regulation and reaction mechanism of the carbamyl phosphate synthetase and aspartate transcarbamylase of bakers' yeast.

Kinetic studies of the carbamyl phosphate synthetase activity (CPSase) of bakers' yeast revealed an absolute requirement for K+ ions ; KM values for two of the substrates, glutamine and bicarbonate, were found to be 5 X 10(-4) M and 3 X 10(-3) M respectively. CPSase activity of the purified enzyme aggregate (M.W. 800,000) was extremely sensitive to UTP with a Ki of 2.4 X 10(-4) M. The purine nucleotide intermediate, XMP, was a strong activator of CPSase, acting at a site different from the regulatory site at which UTP binds ; XMP activation diminished at high concentrations of the substrate Mg-ATP. Studies of the reaction mechanism of CPSase revealed that it involved the sequential addition of the substrates bicarbonate and Mg-ATP, liberation of ADP, addition of glutamine, binding of ATP and then release of ADP and the product carbamyl phosphate. Studies of the reaction mechanism of the aspartate transcarbamylase (ATCase) of the aggregate yielded data which were not compatible with any of the usual models ; whichever reaction mechanism is ultivately found to fit the data, it will probably prove applicable both to the ATCase of the aggregate and to the disaggregated ATCase subunit (MW 138,000).

T-dependent B-cell activation is signalled by an early increase in potassium influx.

When splenic lymphocytes from RNC nu/+ mice were enriched for T cells by lectin purification, mixed to constant cell density with splenocytes from syngeneic nude mice, and cultured with ConA, a proliferative response ensued which was greater than that expected from the T cells alone. This was shown both by incorporation of 3H-thymidine after 48 h of culture and by uptake of potassium (measured as 86Rb) after 15 h. Analysis of metaphase chromosomes stained with Hoechst dye 33258 from co-cultured T-enriched and nude lymphocytes (from female and male donors, respectively) mixed in the proportion 1:4 showed that 40% of the mitotic cells came from nude spleen. About half of the blast cells in such mixtures stained strongly with fluorescein-coupled goat anti-mouse immunoglobulin; T-cell blasts did not stain under these conditions. Treatment of the nu/nu cells with anti-Thy 1.2 and complement had no effect on their subsequent proliferation in coculture. B lymphocytes from nude mouse spleen were therefore activated to proliferate in this system. This B-cell activation can be detected by increased potassium uptake 15 h after the initiation of co-culture. Thus the increased monovalent cation flux (previously demonstrated when B and T lymphocytes were separately stimulated) also occurs when B cells are stimulated through cooperation with mitogen-activated T cells, and is also detectable early in culture. T-dependent activation of B cells is therefore detectable considerably earlier than by conventional assays (such as plaque formation).

Stimulation of the murine mixed leukocyte reaction: optimal proliferation of responders requires activation of stimulators.

In characterizing the functional properties of the stimulators in a mixed leukocyte reaction (MLR) we have established that the optimally effective stimulators must themselves be capable of undergoing the early stages of proliferative activation. We treated the stimulators with sublytic doses of a variety of agents (ouabain, ultra-violet radiation, 5-fluorouracil and colchicine), which have been reported to inhibit their stimulating capacity, and then in parallel experiments, we examined the treated cells for their capacity to stimulate allogeneic cells and their ability to respond to Con A. A direct correlation was found between the capacity of mitomycin C-treated splenocytes to stimulate allogeneic cells in an MLR and their ability to undergo changes characteristic of pre-S phase activation, namely, increases in K+ influx and in cell size, in response to Con A. This correlation also existed in the case of metabolically active but immunosuppressed splenocytes from mice undergoing late graft-versus-host (GVH) reaction: these cells did not show increase in potassium influx in response to Con A and their stimulating ability was inhibited. Using fluorescein labelled anti-Thy 1.2 as a marker for T cells in the stimulating population, it was shown that a considerable fraction (up to one third) of the blasts in a mixed leukocyte culture (MLC) originated in the mitomycin C-treated stimulating population. These data are consistent with the hypothesis that induction of maximal proliferation, in MLR, requires that certain of the cells (mainly T cells) of the stimulating population undergo the early stages of activation.

Rapid rejoining of DNA strand breaks in resting human lymphocytes after irradiation by low doses of 60Co gamma rays or 14.6-MeV neutrons.

The production and repair of DNA strand breaks was studied in human lymphocytes by means of a sensitive fluorometric technique. Lymphocytes were isolated by conventional methods and air-equilibrated suspensions were irradiated by low doses (less than or equal to 2 Gy) of either 60Co gamma rays or 14.6-MeV neutrons at 0 degree C. The apparent yield of initial strand breaks induced by neutrons was only 36% of that induced by gamma rays, in agreement with the observations of other workers. Resting lymphocytes were found to be proficient in their ability to rejoin gamma-induced strand breaks at 37 degrees C; rejoining followed biphasic kinetics, with 70% of the breaks disappearing with a half-life of about 3 min. Although the initial number of breaks induced by neutron irradiation was low, after 20 min of incubation the residual number of breaks was very similar for the two forms of radiation.

Bilateral burning foot pain: monitoring of pain, sensation, and autonomic function during successful treatment with sympathetic blockade.

We describe a patient with burning pain in both feet associated with local autonomic disturbances following bilateral traumatic sciatic mononeuropathies. The diagnosis of a sympathetically maintained pain was confirmed through a prompt response to sympathetic blockade. Although a mild alcohol-nutritional neuropathy was found, the clinical findings strongly suggested a diagnosis of bilateral causalgia. Clinical evaluation and quantitative sensory testing were performed prior to and after successive unilateral lumbar sympathetic nerve blocks. After unilateral blockade, bilateral improvement was recorded in measures of pain, sudomotor function, and foot temperature. Other measures of autonomic function showed variable responses to sympathetic blockade. Quantitative sensory testing revealed a dramatic alteration in the contralateral limb's thermal sense following unilateral block. This case underscores the potential for bilateral causalgia and provides additional evidence for a central mechanism operating in this disorder. The relationship between bilateral causalgia and the "burning feet syndrome" in alcoholic neuropathy is discussed.

The effect of tin chloride on the structure and function of DNA in human white blood cells.

Tin compounds are being used increasingly in the home, in industry and in medicine. There have been relatively few studies on the long term biological effects of this metal, although acute effects have been documented. In this report we describe experiments which show that tin(II), as stannous chloride, is readily taken up by human white blood cells (WBC) and can cause damage to DNA. Damage was detected in WBC after exposure to 10-50 microM tin(II) for 30 min at either 0 degree or 37 degrees C. The amount of damage observed was more extensive than that produced by exposure of cells to equimolar amounts of chromium(VI), a known carcinogen and DNA damaging agent. Additional indication of cellular damage is that exposure of human lymphocytes or mouse splenocytes to tin(II) interfered with their ability to be stimulated by the polyvalent mitogen concanavalin A (Con A). By contrast, tin(IV) was not taken up by cells, did not cause DNA damage nor did it inhibit stimulation of DNA synthesis in cells that were exposed to Con A.

The effect of stannous and stannic (tin) chloride on DNA in Chinese hamster ovary cells.

Tin(II) at concentrations up to 500 microM stannous chloride (SnCl2), produced extensive DNA damage, as detected by alkaline sucrose gradient (ASG) analysis in Chinese hamster ovary (CHO) cells treated for 1 h at 37 degrees C in serum-free minimal essential medium (MEM). However treatment of cells with tin(IV), as stannic chloride (SnCl4), produced no such DNA damage. There was no loss in colony formation 6 days after either treatment suggesting that the DNA damage induced by the tin(II) was rapidly repaired and/or that DNA synthesis proceeded on the damaged templates permitting cell division to occur. Alternatively, the type of DNA damage caused by tin(II) may not lead to a reduction in colony-forming ability. Tin(II) produced about 200 times more ASG detectable DNA damage on an equi-molar basis than did Cr(VI), a known human carcinogen. This study indicates that tin(II) may be potentially genotoxic.