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Acid-base transporters of rabbit and porcine conjunctival epithelia were identified and localized with immunoblotting and immunohistochemical techniques using specific antibodies against carriers commonly found in epithelia, i.e. the Cl(-)/HCO3(-)exchanger (AE2), Na(+)/H(+) exchanger (NHE-1, -2, -3) and the electrogenic Na(+)-(n)HCO3(-) cotransporter (NBC). Western blot analysis demonstrated that anti-AE2 reacted with an approximate 170 kDa protein in both rabbit and pig cell membranes prepared from separately isolated bulbar and palpebral conjunctivae. NHE1 was similarly identified in these distinct conjunctival regions but results with anti-NBC were ambiguous. Histochemical examinations indicated that the AE2 and NHE1 proteins reside on the basolateral surfaces of the plasma membrane throughout the multilayered tissue. The immunostaining of porcine cryosections for AE2 and rabbit sections for NHE1 was specific, because of its abolishment following either pre-absorption with the corresponding peptide or omission of the primary antibody. Screening with anti-NBC produced weak staining of the sections that appeared to be non-specific. For confirmation of these results, the acid-base transporters present in rabbit cell cultures of conjunctival epithelia were ascertained from the changes in intracellular pH (pH(i)) evoked upon sequential superfusion with media of altered composition. This approach readily obtained Na(+)- and Cl(-)-dependent pH(i)effects consistent with the existence of Cl(-)/HCO3(-) and Na(+)/H(+)exchange activities. Evidence for the presence of NBC could not be acquired, thereby substantiating the observations from the immunodetection techniques. The identity and location of the antiporters that were found suggested that these elements could contribute to transcellular Cl(-)transport in the basolateral-to-apical direction. To test this possibility, the effects of AE and/or NHE inhibition were determined on the bumetanide-insensitive Cl(-)-dependent short-circuit current across rabbit conjunctivae freshly isolated in Ussing-type chambers. These experiments revealed that such current is indeed sustained by the antiporters. Results with acetazolamide further suggested that the contribution of the acid-base transporters towards transepithelial Cl(-)secretion is variable and dependent upon individual rates of metabolic CO(2)production. Overall, the present study provides an initial identification of the acid-base transporters present in the conjunctiva. Besides their likely role in intracellular pH regulation, the parallel, basolateral expression of AE2 and NHE1 indicates that these elements do not directly contribute to the pH of the tear film but may complement the Na(+)-2Cl(-)-K(+)cotransporter in effectuating Cl(-)secretion., (Copyright 2001 Academic Press.)

The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical properties of the short-circuited rabbit conjunctiva were examined. With this epithelium, the short-circuit current (I(sc)) measures Cl(-) secretion plus an amiloride-resistant Na(+) absorptive process. Apical addition of 5-HT (10 microM) elicited a prompt I(sc) reduction from 14.2 +/- 1.2 to 10.9 +/- 1.2 microA/cm(2) and increased transepithelial resistance from 0.89 +/- 0.05 to 1.03 +/- 0.06 kOmega. cm(2) (means +/- SE, n = 21, P<0.05). Similar changes were obtained with conjunctivae bathed without Na(+) in the apical bath, as well as with conjunctivae preexposed to bumetanide with the Cl(-)-dependent I(sc) sustained by the parallel activities of basolateral Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. In contrast, the 5-HT-evoked effects were attenuated by the absence of Cl(-) (DeltaI(sc) = -0.5 +/- 0.2, n = 5), suggesting that reduced Cl(-) conductance(s) is an effect of 5-HT exposure. In amphotericin B-treated conjunctiva and in the presence of a transepithelial K(+) gradient, 5-HT addition reduced K(+) diffusion across the preparation by 13% and increased transepithelial resistance by 4% (n = 6, P < 0.05), indicating that an inhibition in K(+) conductance(s) was also detectable. Significant electrical responses also occurred under physiological conditions when 5-HT was introduced to epithelia pretreated with adrenergic agonists or protein kinase C, phospholipase C, phosphodiesterase, or adenylyl cyclase inhibitors or after perturbation of Ca(2+) homeostasis. Briefly, the conjunctiva harbors the only known Cl(-)-secreting epithelium in which 5-HT evokes Cl(-) transport inhibition; receptor subtype and signal transduction mechanism were not determined.

Purpose: To investigate possible regional expression of transport systems in the conjunctival epithelium given distinct differences in morphological appearance between the bulbar and palpebral epithelia as well as variations found in the proportions of Na absorptive versus Cl secretory activities in electrophysiological studies., Methods: Mouse monoclonal antibodies against the alpha1-subunit of Na-K-ATPase and Na-K-Cl cotransporter (NKCC1) and a rabbit polyclonal antibody against the Na-glucose cotransporter (SGLT1) were used in immunoblotting and immunofluorescent labeling of frozen fixed sections isolated from either the bulbar and palpebral regions of the conjunctiva., Results: Western blot analysis clearly demonstrated the presence of Na-K-ATPase, NKCC1 and SGLT1 proteins in both bulbar and palpebral conjunctiva. Indirect immunofluorescence studies on bulbar and palpebral conjunctival portions revealed intense staining by the anti-NKCC1 and anti-alpha1-Na-K-ATPase antibodies exclusively at the basolateral surfaces, whereas the anti-SGLT1 antibody was used with porcine conjunctiva to elicit strong and unambiguous staining along the apical plasma membrane., Conclusions: Proteins that mediate the transport activities of the Na-K-ATPase and Na-K-Cl cotransporter are uniformly distributed throughout the palpebral and bulbar regions of the rabbit conjunctival epithelium. Although the Na-glucose cotransporter could be detected in immunoblots of the rabbit, this cotransporter appears to be uniformly distributed as well, based upon immunohistochemical sections of the pig conjunctiva. Thus, it appears likely that mechanisms for Cl secretion and Na absorption reside in both bulbar and palpebral segments of the conjunctival epithelium.

Corneal stromal hydration is maintained by an active HCO3- transport mechanism located in the corneal endothelium. Whilst modulation of transport activity by changes in intracellular cAMP concentration have been noted, the site of effect is undefined. To resolve this question, the effects of Rolipram, a cAMP phosphodiesterase inhibitor, on endothelial physiology were determined. Addition of 0.1 mM Rolipram caused a threefold increase in intracellular cAMP with no change in cGMP. Associated with the increase in cAMP was a transient whole corneal thinning and a similarly transient increase in trans-endothelial potential difference, short-circuit current and resistance. The membrane potential hyperpolarized and the intracellular Na+ concentration decreased. The decreased intracellular Na+ was associated with an increased rate of Na+ extrusion between the endothelial cell and extracellular space. It is concluded that Rolipram increases the concentration of cAMP which activates the basolateral membrane Na+/K+-ATPase activity and increases net HCO3- transport. In addition there is a reduction in endothelial permeability which combined with the increase in pump activity may jointly explain the observed stromal thinning. The duplicity of responses indicates that if cAMP has a physiological role in regulating corneal hydration then it may operate on both the endothelial pump and the endothelial permeability.

The regulation of Na(+)transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl(-)secretion and Na(+)absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl(-)free media. In this situation, the short-circuit current (I(sc)) measures an amiloride-resistant Na(+)absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 microm) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na(+)-dependent I(sc)by approximately 3.5-4.5 microA cm(-2)(minimal 40% increase) and reduced transepithelial resistance (R(t)) by at least 7% (P<0.05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 microm), which in itself inhibited the I(sc)by 0. 5 microA cm(-2), attenuated the I(sc)responses of the cAMP-elevating agents (P< 0.05, unpaired data). In reverse, H-89 promptly decreased the I(sc)by 1.5-2.5 microA cm(-2)and increased R(t)by 5% (P<0.05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I(sc)by 12 and 22% respectively over baseline and reduced R(t)by 6% (P<0.05). Similarly, in the presence of a transepithelial K(+)gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1.8 microA cm(-2)and decreased the R(t)by 4% (P<0.05), changes that were reversed by subsequent H-89 addition. Under Cl(-)rich conditions, pretreatment with 5 m m Ba(2+)reduced the basal I(sc)by 59% and blocked the cAMP-induced I(sc)stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba(2+)-inhibitable (voltage insensitive) basolateral K(+)conductance in rabbit conjunctival epithelial cells. The action of Cl(-)secretogogues acting via cAMP on basolateral K(+)channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl(-)secretion and Na(+)absorption in the conjunctiva., (Copyright 2000 Academic Press.)

Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial transporters. The present work examined the influence of phorbol esters, which are known activators of PKC isoenzymes, on the short-circuit current (Isc), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the Iscmeasures a Na+-dependent, bumetanide-inhibitable Cl-transport in the basolateral-to-apical direction plus an amiloride-resistant Na+absorptive process in the opposite direction. Additions of phorbol 12-myristate-13-acetate (PMA) to the basolateral bathing media did not affect the transepithelial electrical parameters; but its introduction to the apical bath at 1 and 10 micrometers elicited a transient ( approximately 2 min duration) Iscspike followed by a sustained reduction relative to the control level. Such PMA-elicited Iscreductions were from 14. 0+/-2.0 to 3.1+/-0.8 microA cm-2(+/-s.e.m.'s, n =3) at 1 micrometer and from 16.5+/-1.9 to 4.6+/-0.7 microA cm-2(n =22) at 10 micrometers. The former concentration failed to produce extensive Iscreductions in 3 other experiments. Similar results were obtained with phorbol 12, 13-dibutyrate (PDBu). Its apical administration at 0.1 micrometer reduced the Iscfrom 18.5+/-4.1 to 7.8+/-2.0 (n =3), and from 16. 5+/-2.9 to 6.9+/-1.2 (n =7) when introduced at 1 micrometer. The phorbol-evoked Iscreductions occurred without a simultaneous change in transepithelial resistance (Rt). However, after about 15-20 min, Rtgradually declined by about 25%. In contrast to these results, treatment with a phorbol ester known not to activate PKC (4-alpha-PMA) did not affect the electrical parameters when added at 10 micrometers. PMA- and PDBu-evoked Iscreductions could be obtained with conjunctiva that were (1) pretreated with bumetanide, (2) bathed in Cl--free media, and (3) pretreated with amphotericin B, changes consistent with a likely inhibition of the basolateral Na+/K+pump. Such Iscinhibitions were attenuated with conjunctiva pre-exposed to 1 micrometer staurosporine, a nonselective kinase inhibitor known to suppress PKC activity. Staurosporine, in itself, produced a rapid 26% Iscinhibition (n =15) along with a 17% Rtincrease upon its apical introduction. These electrical responses were less extensive in Cl--free media and absent in amphotericin B-treated conjunctiva, suggesting the presence of a kinase-mediated regulation of apical channels for both Na+and Cl-. Overall, these results imply that in addition to previously demonstrated epinephrine-elicited, up-regulation of Cl-secretion, mechanisms may also exist, via PKC activation, to suppress Na+/K+pumping and consequently reduce transepithelial transport rates., (Copyright 1999 Academic Press.)

Identification of Rhizoctonia solani, R. oryzae and R. oryzae-sativae, components of the rice sheath disease complex, is extremely difficult and often inaccurate and as a result may hinder the success of extensive breeding programmes throughout Asia. In this study, primers designed from unique regions within the rDNA internal transcribed spacers have been used to develop a rapid PCR-based diagnostic test to provide an accurate identification of the species on rice. Tests on the specificity of the primers concerned showed that they provide the means for accurate identification of the Rhizoctonia species responsible for sheath diseases in rice.

Amiloride (0.5 mM) inhibited the rate of entry of Na+ into corneal endothelial cells by more than half ((0.76 +/- 0.10) to (0.21 +/- 0.10) microEq cm(-2)h(-1)). The same concentration of amiloride caused only minimal disturbance to corneal hydration control by the endothelium (range 0-12%). Amiloride (0.5 mM) and acetazolamide (1 mM) reversibly inhibited trans-endothelial short circuit current by about a half. Their combined effect was not additive. Acetazolamide (1 mM) reduced net HCO3- flux across the short-circuited endothelium by about the same amount ((0.50 +/- 0.11) microEq cm(-2)h(-1)) that amiloride (0.5 mM) reduced Na+ entry into the cells ((0.55 +/- 0.14) microEq cm(-2)h(-1)). Low concentrations of amiloride (10 microM) had little effect on the transport characteristics of the endothelium, indicating that Na+ entry into the endothelial cells under physiological conditions is not primarily through Na+ channels. The data are consistent with an Na+/H+ exchanger acting in tandem with carbonic anhydrase through a pathway which could have a regulatory role on endothelial transport via its effect on Na+ re-entry. Residual trans-endothelial HCO3- transport, apparently unaffected by amiloride or acetazolamide inhibition, is calculated to be of sufficient magnitude to maintain corneal hydration.

This article comments on several of the major issues facing military optometry which were aired earlier this year at the AOA Federal Service Optometry Seminar. Among these issues, all of which influence recruitment and retention of optometry officers, are constructive credit, bonus and incentive pay, promotion policies, and optimum scope of care. Dr. Turner emphasizes that solutions to these problem areas depend not only upon AOA actions but also personal efforts and professionalism of military OD's.

A passage line of a spontaneous hamster fibrosarcoma is contaminated by the virus. of lymphocytic choriomeningitis. Tumors from animals receiving implants when newborn contain high titers of infectious lymphocytic-choriomeningitis virus and complement-fixing antigen, and hamsters receiving implants when weanlings develop high titers of complement-fixing antibody against lymphocytic-choriomeningitis virus. In contrast with the specific reactions of tumorous hamsters to the initiating virus in virus-induced tumors, the development of complement-fixing antibody to lymphocytic-choriomeningitis virus does not depend on the development of tumors. Infant hamsters bearing the tumor have a generalized subclinical infection and seem able to spread virus to other hamsters and to humans.

The induction of lymphomas in C57BL mice by methylcholanthrene, urethan, or diethylnitrosamine was accompanied by the development of murine leukemia viral antigen in most of the lymphoid tumors. The cell-free transmission of lymphomas induced by methylcholanthrene and the development of antibody to murine leukemia virus prior to the detection of overt lymphoma in these mice suggest that unmasking of a latent leukemia virus is an indigenous actuating cause of the lymphomas.

Taylor-Robinson, David (National Institutes of Health, Bethesda, Md.), Norman L. Somerson, Horace C. Turner, and Robert M. Chanock. Serological relationships among human mycoplasmas as shown by complement-fixation and gel diffusion. J. Bacteriol. 85:1261-1273. 1963.-Antigenic relationships among human mycoplasmas were studied by complement-fixation and agar gel diffusion techniques. Four recognized human species Mycoplasma hominis type 1, M. hominis type 2, M. salivarium, and M. fermentans were antigenically distinct in these tests. In addition, M. pneumoniae (Eaton agent, the etiological agent of cold agglutinin-positive atypical pneumonia) was different from these four species. Although these species were distinct, evidence of shared antigenic components was obtained in complement-fixation and agar gel diffusion tests. Since rabbits were immunized with mycoplasmas grown in rabbit muscle infusion broth supplemented with rabbit serum or, in the case of M. pneumoniae, with infected chick embryo lung suspension, the possibility that the heterologous reactions resulted from antibody to growth medium components could be excluded. Four recent mycoplasma isolates from the oropharynx were analyzed, and three were shown to be closely related to M. hominis type 1, and the fourth was closely related to M. salivarium. Although the recent isolates could not be distinguished from the related "prototype" human species by complement-fixation, differences could be detected by the agar gel diffusion technique.

Tests for the group-specific antigen of the C-type RNA tumor virus showed that mouse embryos of all strains tested, at some stage of development in utero, revealed detectable titers of group-specific antigen in one or more of their tissues; younger, rather than older, embryos were likely to be positive, particularly in those strains which normally reveal little or no expression of the RNA genome postnatally. The antigens were found in embryos of low-leukemia strains, free of infectious virus. These new findings support a previously stated hypothesis that the genome of RNA tumor viruses, mostly switched off for infectious virus expression, is vertically transmitted as part of the natural genetic apparatus of normal mouse cells. Since group-specific antigens have also been described in chick embryos and immunological tolerance to homologous group-specific antigens has been demonstrated in hamsters and cats as well as in mice and chickens, the hypothesis has been extended to include vertebrate cells in general. Finally, the high incidence and titers of the group-specific antigen suggest that the genes for RNA tumor virus, which later in life act as determinants of cancer, may be important also as gene determinants in the developing embryo.