Frequency of antibody to a virus-induced tumor-associated antigen (AG-4) in Japanese sera from patients with cervical cancer and controls. Microquantitative complement fixation was used to detect antibody to AG-4, a tumor-associated herpesvirus type 2-induced antigen, in sera from Japanese patients with cervical neoplasia. The prevalence of antibody to AG-4 in cervical cancer patients is 47% as compared to 7% in control women. That AG-4 antibody is less prevalent in Japanese than in American cancer patients reflects the frequency of genital herpesvirus type 2 infections in Japan. These findings confirm the association of AG-4 with cervical cancer in a proportion of Japanese patients with neoplastic disease; they implicate factors other than herpesvirus type 2 in another proportion of cervical cancer patients. The possibility that herpesvirus type 1 is one of these factors is considered.


Methylation analysis of the carbohydrate portion of carcinoembryonic antigen. The carbohydrate structural units of carcinoembryonic antigen samples isolated from four different tumors were quantitated using gas chromatography-mass spectrometery after methylation and subsequent conversion to their alditol acetates. Different carcinoembryonic antigen preparations showed some quantitative but no qualitative differences in the structural units present. The results indicate that a large portion of the fucose residues in the glycoprotein were linked to N-acetylglucosamine and that most of the branching mannose residues were probably linked to three N-acetylglucosamine residues.


Resistance of human melanoma cells to ultraviolet radiation. A series of five human melanoma cell lines has been demonstrated to be highly resistant to ultraviolet (UV) radiation, with a D0 of 400 ergs/sq mm. Melanotic melanoma cells were found to increase their production of melanin following UV radiation, whereas some amelanotic cells did not. Melanotic and amelanotic melanoma cell lines exhibited the same UV resistance; melanoma and nonmelanoma cells formed the same numbers of thymine dimers at a given UV dose. These data imply that melanin does not play a major role in protecting DNA of melanoma cells against UV damage in culture. The rates of removal of thymine dimers from DNA of melanoma cells were comparable to those in UV-sensitive, nonmelanoma cell lines, so that rapid excision repair does not explain UV resistnace in the melanoma cells. No DNA strand breakage was detected in a melanoma cell line at moderate UV doses.


Transplantation resistance to mouse ependymoblastoma following immunization with dehistonized syngeneic tumor chromatin. Female C57BL/J6 mice pretreated with dehistonized ependymoblastoma chromatin rejected 5 x 10(4) or 10(5) s.c.-implanted syngeneic ependymoblastoma cells. Control mice that received Freund's adjuvant containing dehistonized chromatin from normal mouse brain developed palpable tumor nodules in 90% and 35 to 45% of the animals, respectively. Increased binding of spleen lymphocytes from mice pretreated with dehistonized tumor chromatin to Sepharose 4B coupled with components of dehistonized tumor chromatin was observed. This provides initial evidence for the presence of "committed" lymphocytes in mice pretreated with dehistonized chromatin from this mouse ependymoblastoma.


The differential effects of chemical carcinogens on vitamin A status and on microsomal drug metabolism in normal and vitamin A-deficient rats. Male Sprague-Dawley rats were maintained on a vitamin A-deficient diet for 5 weeks. Although serum and hepatic levels of vitamin A were significantly lower at this time, no outward signs of vitamin A deficiency were present. Hepatic microsomal levels of cytochrome P-450 in the vitamin A-deficient animals were 70% that of the control animals. Of the three microsomal enzymes studied, ethylmorphine N-demethylase, aniline hydroxylase, and aminopyrine N-demethylase, only the last one was adversely affected by vitamin A deficiency. 3-Methylcholanthrene, phenobarbital, and 2-acetylaminofluorene had a greater inductive effect and cytochrome P-450 in vitamin A-deficient rats. 4-Dimethylaminoazobenzene treatment decreased in the level of cytochrome P-450 in control rats more than in deficieny rats. The hepatic concentration of vitamin A was significantly reduced in control rats that were given injections of 3-methylcholanthrene, 2-acetylaminofluorene, or phenobarbital. Benzo(a)pyrene and 4-dimethylaminoazobenzene had less effect.


Development of an assay for aryl hydrocarbon (benzo(a)pyrene) hydroxylase in human peripheral blood monocytes. An assay has been developed and measurements of aryl hydrocarbon [benzo(a)pyrene] hydroxylase have been made in peripheral blood monocytes from a human population. Treatment with benz(a)anthracene in cell culture increased aryl hydrocarbon hydroxylase activity from 6.5 to 37-fold in monocytes from each of 25 apparently healthy donors. A weak correlation (r = 0.38) was observed between the induction ratios obtained with monocytes and lymphocytes from the same donors. Reproducibilities of the monocyte and lymphocyte assays were comparable. In monocytes, the measurement of basal and induced aryl hydrocarbon hydroxylases activity does not require pretreatment with mitogens as is the case with lymphocytes. Monocytes also exhibit a much wider range of induction ratios than do lymphocytes.


The effect of adriamycin on the cell cycle traverse of a human lymphoid cell line. The kinetic response of a human lymphoma cell line to adriamycin was analyzed by means of pulse cytophotometry. Depending on concentration and exposure time, cell cycle progression was delayed in G1, S, and G2 phases. There was a differential sensitivity for the interaction of adriamycin with the transit through these phases. G2 arrest could be induced by low concentrations of adriamycin, whereas the block in G1 was exerted only after long-term treatment with high concentrations and was completely reversible after drug removal. Delay in S-phase transit was transient in spite of continuous exposure to high concentrations of adriamycin. Thus, concentration and duration of treatment determined the magnitude of G2 arrest as well as onset and rate of G2 accumulation due to progression delay in G1 and S phases. Cell age had only little influence on the degree of subsequent G2 arrest. Irreversibility of the G2 block strongly suggests eventual cell death in G2 phase, which may be utilized as a predictive test for response to adriamycin in vivo.


Influence of glucocorticoid, estrogen, and androgen hormones on transformation of human cells in vitro by feline sarcoma virus. Infection of human foreskin cells (D-550) by the Snyder-Theilen strain of feline sarcoma virus produced small but countable foci and demonstrated "single-hit" dose-response kinetics. Significant quantitative and qualitative enhancement of focus formation was observed when the glucocorticoid hormones, dexamethasone, hydrocortisone, cortisol acetate, and prednisolone were added to cell cultures (1.0 mug/ml) 24 hr postinfection. However, aldosterone, while inducing qualitatively larger foci, did not bring about a quantitative enhancement in total foci number. By contrast, 17beta-estradiol, progesterone, cortisone acetate, methyltestosterone, and estrone elicited little or no effect on focus induction by Snyder-Theilen feline sarcoma virus. Evidence is suggestive of a posttranscriptional effect possibly modulating viral genome expression resulting in an increased efficiency of viral transformation, and an increased proliferation of transformed cells.


Electrocardiographic and pathological studies of the heart in experimental guinea pig leukemia. Electrocardiographic tracings were performed on 26 strain 2 guinea pigs in which leukemia was induced by inoculation of L2C leukemic cells. All inoculated animals developed leukemia. In terminal phases of this disease significant electrocardiographic changes were observed in 20 of the 26 animals; in 5 animals the electrocardiograms were normal and in 1 guinea pig the changes were borderline. The most significant changes consisted of the onset of a Q-wave or a T-wave inversion or both. Pathological examination of the heart removed in the terminal phase of the disease revealed infiltration of the endocardium and epicardium and in the capillaries of the myocardium. Such areas of infiltration, when viewed with the electron microscope, revealed the presence of leukemic cells within the lumen of capillaries and in areas immediately surrounding the capillaries. Infiltration of myocardial fibers was not observed. The electrocardiographic changes observed in guinea pig leukemia may be related to the leukemic infiltration of myocardial capillaries and the resulting anoxia.


Growth support of small B16 melanoma implants with nitrosourea-sterilized fractions of the same tumor. B16 melanoma cells sterilized in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea or in vivo with trans-1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, have been used to enhance the percentage of tumor takes with small s.c. implants of viable cells and to reduce the latent period between tumor implantation and palpability. The admixture of trans-1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea-inactivated cells with viable cell implants reduced the number of cells required to produce tumors in 50% of the animals by approximately 3 log10 units and markedly reduced the time of tumor appearance from implants of up to 10(6) cells. Similar results were obtained with 1,3-bis(2-chloroethyl)-1-nitrosourea-sterilized cells. The growth-supporting effect obtained with the nitrosourea-inactivated cells appeared to be as pronounced as that previously reported, for this tumor system, with radiation-inactivated cells.


Specific potentiation of L1210 vaccine by pyran copolymer. Pyran copolymer (NSC 46015) was found to potentiate strongly the immune response of C57BL/6J X DBA/2 F1 mice to 10(4) live L1210 tumor cells following suboptimal vaccination with 10(7) radiation-inactivated L1210 cells. Optimal immunity to challenge was produced by concomitant i.p administration of pyran and L1210 vaccine, and activity was dependent upon both pyran and vaccine dosages. In addition, this immunopotentiation seemed to be related to the intrinsic viscosity of different pyran preparations tested, although all the pyran compounds had significant activity. Furthermore, the increased immunity of subsequent live tumor challenge appeared to be specific for the vaccinating cell type.


Decreased lymphocyte adenosine deaminase activity in tumor patients. Adenosine deaminase has been measured in the lymphocytes of individuals with various types of solid tumors. The mean activity was found to be significantly lower when compared with control individuals of a similar age.


Thymic hormone modulation of leukemogenic virus replication. The effect of purified calf thymus extract, thymosin, on infection with murine leukemia virus (MuLV) (Rauscher) was studied in adult thymectomized BALB/c mice. The course of infection was determined by virus titer in the blood plasma, by enumeration of spleen cells replicating MuLV (i.e., infectious centers), and by the examination of cells for expression of virus-induced cell membrane antigen. Thymectomy (performed 1 week prior to the virus injection) decreased MuLV infection by all three parameters. However, administration of thymosin (500 mug, given in equal doses 2 days before and at the time of injection of virus) caused a marked increase of plasma titer of virus, as well as an increased number of spleen cells generating MuLV as compared to controls treated either with purified spleen fraction or with diluent (buffered salt solution). More than two doses of thymic extract (up to nine, given every 2 days after infection) had no further effect on plasma virus titer or number of splenic infectious centers. Furthermore, treatment of mice with either thymic or splenic extracts increased the incidence of virus related membrane antigen appearing on the surface of spleen cells, as compared to diluent-treated control animals. These results indicate that thymosin may enhance infection by or replication of murine oncornaviruses.


Bioassay for quantitating circulating tumor cells in a syngeneic mouse tumor system. A bioassay is described for the quantitation of tumor cells in blood specimens in a syngeneic mouse tumor system (Sarcoma 1 in A/J mice). The procedure involved i.m. injection of blood containing tumor cells into each thigh of normal recipient mice and, 14 days later, examination of the sites of injection for evidence of tumor growth. For each specimen, a tumor index was calculated based on the number of tumor takes and the size of the tumors. The number of tumor cells was determined by comparison with tumor indices from standard specimens with known number of tumor cells. Optimal conditions for this assay were investigated. We have used this bioassay to quantitate tumor cells in the venous blood of tumor-bearing animals under various treatments and manipulations. At the same time, the incidence of regional node metastasis was obtained by direct histological examination. Surgical removal of a well-established primary tumor enhanced the dissemination of the tumor, as evidenced by an increased incidence in regional node metastasis and an increase in the number of tumor cells reaching the venous circulation. Similar results were obtained when the tumor-bearing feet were ligated to produce ischemia of the primary tumor. Repeated physical trauma to the primary tumor resulted in increased dissemination of tumor cells into the venous circulation, but it did not increase the incidence of regional node metastasis. Immunosuppression of the tumor-bearing animals increased the dissemination of tumor cells, whereas immunostimulation decreased the dissemination.


Tumorigenicity in vivo and induction of malignant transformation and mutagenesis in cell cultures by adriamycin and daunomycin. The two anthracycline antitumor antibiotics, adriamycin and daunomycin, have been tested for tumorigenic activity, and the results confirm previous findings that they can induce mammary tumors in female rats receiving single i.v. doses. Both substances are highly potent in producing malignant transformation and mutation in mammalian cell systems in vitro. Their transformming activity is comparable to that of the potent carcinogen, N-methyl-N'-nitro-N-nitro-soguanidine. Actinomycin D, although similar to the anthracyclines in having high binding affinity for DNA, is only minimally effective in the same in vitro systems and its direct carcinogenic activity in vivo is moot. These results suggest that satisfactory correlations may be obtainable between tests for tumorigenicity in vivo, and assays for transformation and mutagenesis in vitro, and that adriamycin, and daunomycin may have carcinogenic potential in man.


Nonrandom nature of in vivo methylation of dimethylnitrosamine and the subsequent removal of methylated products from rat liver chromatin DNA. This investigation was designed to study whether methylation of liver chromatin DNA by dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-digestible and nondigestible material 4 hr following the administration of [3H]DMN (0.5 mg/250 muCi/100 g body weight). Digestion of such methylated liver chromatin with pancreatic DNase I or micrococcal nuclease and analysis of nuclease-digested acid-soluble products revealed a discrepancy between the radioactivity released (72%) and the nucleotides released (50%) as measured by the absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of both the radioactivity and the absorbance at 260 nm were identical when the total purified DNA isolated from methylated chromatin was used as the substrate instead of chromatin DNA in the nuclease reaction. These results, together with the fact that guanine contents of the DNA of the two fractions of the chromatin isolated by nuclease digestion were identical, suggest that methylation of the nuclease-accessible region of hepatic chromatin DNA is relatively greater than that of the inaccessible region. The study of the removal of methylated products in the accessible region of the chromatin DNA further reveals that, of the methylated products present at 4 hr, 62% is lost by 3 days, 87% is lost by 1 week and 94% is lost by 2 weeks. However, loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 week, and 86% by 2 weeks, thereby suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nuclease-accessible region of chromatin-DNA. The results of this study thus indicated (a) an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and (b) decreased methylation and slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. It is concluded that the distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.


Evaluation of Papanicolaou smear and effect of sample biopsy in follow-up of cervical dysplasia. In a prospective study of women with dysplasia, a superficial sampling biopsy of the cervix was carried out as a periodic check of the cytological findings. The relationship of smear and biopsy results was evaluated, as well as the effect of the biopsy procedure on the subsequent course of dysplasia. We found sufficiently close agreement between smear and biopsy results to conclude that smears and biopsies measure similar aspects of dysplasia. It would appear unwise, however, to rely on Papanicolaou smears exclusively in following women with a history of dysplasia, since negative smears in such women may occasionally show dysplasia in the corresponding biopsy. Periodic corroborative biopsy procedures are therefore indicated in the follow-up care of women with a history of dysplasia. There was no evidence that a superficial sampling biopsy significantly altered the short-term course of dysplasia. There was also no evidence of a cumulative effect of repeated sampling biopsies. These results do not rule out possible effects of other forms of biopsy procedures and schedules on the subsequent course of dysplasia.


Failure of amygdalin to arrest B16 melanoma and BW5147 AKR leukemia. Parenteral amygdalin was found to be ineffective in C57BL/6 mice with B16 melanoma and in AKR mice with BW5147 lymphatic leukemia, in doses ranging from 50 to 5000 mg/kg.


Differences in cellular uptake and cytofluorescence of adriamycin and N-trifluoroacetyladriamycin-14-valerate. Adriamycin-specific fluorescence appears slowly in living cells and is localized in nuclei and chromosomes. N-Trifluoroacetyladriamycin-14-valerate, a recently synthesized adriamycin analog, differs from the parent anthracycline in the rapid appearance of its fluorescence in the cytoplasm of living cells and the lack of any fluorescent binding to nuclei and chromosomes.


Modification of growth of neuroblastoma cells in syngeneic mice by aldehyde-treated neuroblastoma cells. Pretreatment of syngeneic strain A mice with aldehyde-fixed neuroblastoma cells (clone NB6R) almost completely protected the mice against challenge with viable NB6R cells. In contrast, tumor growth was enhanced in mice treated with fixed cells after challenge with viable cells.


Effects of microwave-induced local hyperthermia on mammary adenocarcinoma in C3H mice. Microwave-induced hyperthermia was focused locally upon mammary adenocarcinoma implanted in C3H mice when the tumor reached 6 mm in diameter. In 54 treated mice, all tumors diminished in size after the first exposure (43 degrees, 45 min) and disappeared completely following the second treatment. No other effects from heat application were apparent. All the mice in the microwave-treated group survived the whole period of observation (4 months) without any evidence of tumor, while 18 nontreated controls died within 4 weeks after inoculation.


The 13C-N.M.R. spectra of disaccharides of D-glucose, D-galactose, and L-rhamnose as models for immunological polysaccharides. Complete assignments of the 13C-n.m.r. spectra of disaccharides having beta-glycosidic linkages are presented and discussed. The disaccharides of D-glucose, D-galactose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose are model compounds for 13C-n.m.r. studies of immunological polysaccharides. Changing the nature of the reducing glucopyranose rings (D-glucose to L-rhamnose) has no important influence on the chemical shifts of the carbons of the non-reducing glucopyranose ring (D-glucose). The converse is also true: the chemical shifts of the carbons of the reducing glucopyranose ring (L-rhamnose) are not noticeably affected by a change of the non-reducing unit (D-glucose to D-galactose or 2-acetamido-2-deoxy-D-glucose).


Synthesis and rearrangements of D-glucosyl esters of aspartic acid linked through the 1- or 4-carboxyl group. Catalytic hydrogenation of 2,3,4,6-tetra-O-benzyl-1-O-[1-benzyl N-(benzyloxycarbonyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (1alpha) in acetic acid-2-methoxyethanol gave 1-O-(L-beta-aspartyl)alpha-D-glucopyranose (2alpha) contaminated with 2-O-(L-alpha-aspartyl)-D-glucopyranose (8). Evidence that 8 was formed from the 1-oyl isomer of 1alpha, namely 2,3,4,6-tetra-O-benzyl-1-O-[4-benzyl N-(benzyloxycarbonyl)-L-aspart-1-oyl]-alpha-D-glucopyranose (7alpha), via 1 leads to 2 acyl migration, was obtained by submitting the deprotected D-glucosyl ester to successive N-acetylation, esterification, and O-acetylation; the final product was identified as a approximately 4:1 mixture of 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (4alpha) and 1,3,4,6-tetra-O-acetyl-2-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-D-glucopyranose (6) which were also prepared by definitive methods. On the other hand, deprotection of 1beta gave isomerically pure 2beta which was converted into the peracetylated ester derivative 4beta; an explanation for the differences in aglycon isomeric purity of 2alpha and 2beta is given. Hydrogenolysis of 7beta under the above conditions led to intermolecular transesterification with scission of the C-1 ester bond to give 1-(2-methoxyethyl) L-aspartic acid and D-glucose. Catalytic hydrogenation of 7alpha and 7beta, performed in the presence of trifluoroacetic acid, afforded 1-O-(L-alpha-aspartyl)-alpha- and -beta-D-glucopyranoside trifluoroacetate salts (11alpha and 11beta), respectively. The structure of 11beta was established by successive conversion into 2,3,4,6-tetra-O-acetyl-1-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-beta-D-glucopyranose (5beta) which was also prepared by definitive methods. Analogous treatment of 11alpha gave the N-acetyl derivative 12 which underwent 1 leads to 2 acyl migration during esterification with diazomethane to give the N-acetyl methyl ester derivative 10; acetylation of 10 afforded 6.


Synthesis and chemical behaviour of D-glucosyl esters of glutamic acid having the side-chain carboxyl group involved in the glycosidic linkage. Simultaneous and stepwise deprotection of the fully benzylated D-glucosyl esters of 1-benzyl N-benzyloxycarbonyl- and N-tert-butyloxycarbonyl-L-glutamic acid (1 and 5, respectively) have been examined. Catalytic hydrogenation of 1 led to intramolecular aminolysis to give pyroglutamic acid and D-glucose, but similar treatment in the presence of trifluoroacetic acid afforded both anomers of 1-O-(L-gamma-glutamyl)-D-glucopyranose, which were characterized as trifluoroacetates (2alpha and 2beta) and converted into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-glutam-5-oyl]-D-glucopyranose (4) which was also prepared by a definitive method. Hydrogenolysis of 5 gave both anomers of 1-O-[N-(tert-butyloxycarbonyl)-L-gamma-glutamyl]-D-glucopyranose (6), which, upon treatment with trifluoroacetic acid at - 10 degrees, afforded 2alpha and 2beta, respectively. The structure of 6beta was established by its conversion into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-beta-D-glucopyranose (7beta), whereas similar treatment of 6alpha gave a mixture of 1,3,4,6-tetra-O-acetyl-2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (9) and 7alpha. A 1 leads to 2 acyl migration occurred during esterification of the aglycon carboxyl group of 6alpha with diazomethane to give 2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (8).


Comb-like derivatives of amylose having (1-6)-linked malto-oligosaccharide side-chains. The preparation of acetylated glycosyl bromide derivatives of the higher malto-oligosaccharides was studied by using beta-cyclodextrin or linear malto-oligosaccharides of d.p. 6 and 7. The products were treated with 2,3-di-O-phenylcarbamoyl-6O-tritylamylose in the presence of silver perchlorate (reaction A), and with 2,3-di-O-phenylcarbamoylamylose in the presence of mercuric cyanide and mercuric bromide (reaction B). After removal of the substituents, the branched molecules were characterized by their iodine-binding properties, beta-amylolytic degradation, and their priming activity in phosphorolytic synthesis. The distance between the branch points of the backbone chain was 25-55 and 100-150 units in the products from reactions A and B, respectively. Thus, the frequency of branching was considerably lower than in the comb-like molecules having D-glucose side-chains previously described.


Carbohydrate-binding properties of an immobilized alpha-D-galactopyranosyl-binding protein (lectin) from the seeds of Bandeiraea simplicifolia. The alpha-D-galactopyranosyl-binding lectin from Bandeiraea simplicifolia has been coupled to cyanogen bromide-activated Sepharose 4B. Using this immobilized system, we have been able to study the interaction of the lectin with model carbohydrate-protein conjugates and polysaccharides, and to reaffirm this protein's carbohydrate-binding specificity. The opportunity for the isolation of biopolymers containing alpha-D-galactopyranosyl end-groups is demonstrated by the single-step purification of a new galactomannan from the seeds of Cassia alata.


Studies on the isothermal crystallization of D-glucose and cellulose oligosaccharides by differential scanning calorimetry. Isothermal crystallization from the glassy state of D-glucose and cellulose oligosaccharides (e.g., cellobiose, cellotriose, and cellotetraose) has been studied by differential scanning calorimetry. The crystallization of amorphous D-glucose and oligosaccharides was very difficult in the absence of traces of water. Amorphous cellobiose and cellotetraose crystallized far more rapidly than amorphous D-glucose and cellotriose. The activation energy for the crystallization of cellobiose and cellotetraose was approximately 10-12 kJ. mol(-1), while that for D-glucose and cellotriose was approximately 1-2 kJ. mol(-1). An odd-even effect seemed to be associated with the crystallization process of these saccharides.


Synthesis and stereochemistry of 6-deoxy-6-halogeno-D-glucofuranose cyclic phosphates. 1, 2-O-Cyclohexylidene- and 1, 2-O-isopropylidene-alpha-D-glucofuranose react with hexa-alkylphosphorous triamides to give the corresponding 3, 5, 6-phosphites. Treatment of the latter compounds with chlorine and bromine affords 1, 2-substituted 6-deoxy-6-halogeno-alpha-D-glucofuranose 3, 5-phosphorohalogenidates. Replacement of halogen at phsophorus by hydroxyl and amino groups has been investigated. Cyclic phosphorohalogenidates isomerize in N, N-dimethylformamide. The stereochemistry of the compounds investigated was established by using 1H- and 13C-n.m.r. data.


The chemical structure of a fragment of Micrococcus lysodeikticus cell-wall. A fragment of Micrococcus lysodeikticus cell-wall obtained by cetylpyridinium recipitation from the nondialyzable portion of the degradation products of egg-white lysozyme was studied by the periodate oxidation and methylation procedures. The fragment consists of a polysaccharide chain composed of about 40 repeating (1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranosyluronic acid)-(1 leads to 6)-O-(alpha-D-glucopyranosyl) residues with D-glucopyranosyl residues at both ends. The alpha-D-glucopyranose residue at the reducing end is linked to a phosphate group that is also linked to C-6 of a 2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl residue of a peptidoglycan chain composed of four repeating (1 leads to 4)-O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl] residues. The peptidoglycan chain has, as nonreducing group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, and, as reducing residue, a 2-acetamido-3-O-(D-1-carboxytheyl)-2-deoxy-beta-D-glucose residue.


Structural studies of the polysaccharide antigen of Eubacterium saburreum, strain 49. The polysaccharide antigen produced by Eubacterium saburreum, strain L 49, is composed of D-glycero-D-galacto-heptose and a new sugar, tentatively identified as 6-deoxy-D-altro-heptose. It contains chains of alternating (1 leads to 3)- and (1 leads to 6)- linked beta-D-glycero-D-galacto-heptopyranosyl residues, the latter being substituted with 6-deoxy-alpha-heptofuranosyl groups at O-3. The polysaccharide further contains 0-acetyl groups, linked to O-7 of part of the heptosyl residues and to O-2 of part of the 6-deoxyheptosyl groups.


The rodent incisor tooth proliferon.

The rodent incisor tooth proliferon. The rodent incisor tooth is the site of five cell populations proliferating in harmony: amelocytes, odontocytes, pulp cells, endothelial cells and the periodontal ligament. Their proliferating regions are located in the apex tip, where the various cells originate. Cells displaced from the tooth origin at the apex toward the periphery, mature to perform their specified function. The proliferative events in the tooth are summarized in a conceptual model of the incisor proliferon. The proliferon is an oriented structure with an origin and periphery. It consists of four basic elements: parenchyma, connective tissue, blood vessels and nerve fibres, all interacting continuously. All four are indispensable in the definition of the proliferon.


DNA turnover and thymidine re-utilization in mouse tissues. Mice were injected intravenously with tritiated thymidine (TdR) and 125I-labeled iododeoxyuridine (IUdR) in low doses to provide a simultaneous labeling of tissue DNA with non-toxic amounts of these two precursors. The total activity per organ and the ratio of the two isotopes was measured in the DNA at various times between 1 and 15 days after the injection. Since TdR from dying cells is re-utilized more effeciently than IUdR from the same cells, more labeled TdR than IUdR was retained in the tissue DNA in these experiments. From the slopes of the regression lines, the true rated of turnover of replicating tissue DNA and the per cent re-utilization of TdR were calculated. Re-utilization of TdR varied from 37 to 60% in the six tissues examined.


The migration of lymphocytes across specialized vascular endothelium. I. The entry of lymphocytes into the isolated mesenteric lymph-node of the rat. The chain of lymph-nodes in the rat mesentery was isolated and the preparation was perfused via cannulae in the superior mesenteric vessels. The perfusate consisted of serum to which labelled lymphocytes had usually been added. The entry of radioactively labelled lymphocytes from the blood vessels into the lymph-nodes was studied by scintillation counting and autoradiography. It was observed that: (1) In the perfused node labelled lymphocytes localized in and around post-capillary venules in the paracortex as they do early after i.v. injection. (2) The number of lymphocytes which entered the node was directly proportional to the concentration in the perfusate over the range studied. The proportion of cells retained in the node varied considerably around a mean of 11% of lymphocytes reaching it. (3) The isolated lymph-node released few if any lymphocytes into the effluent (venous) perfusate. (4) Large lymphocytes migrated into isolated lymph-nodes slightly more readliy than did small lymphocytes. (5) Unlike intact cells isolated lymphocyte membranes did not adhere to specialized vascular endothelium.


A two emulsion autoradiographic technique and the discrimination of the three different types of labelling after double labelling with 3H- and 14C-thymidine. The first part of the paper deals with a two emulsion autoradiographic technique for double labelling experiments with 3H- and 14C-thymidine which permits a clear discrimination of the different types of labelling. In the second part the application of this technique to cell kinetic studies is discussed. Accurate discrimination between the different types of labelling, namely purely 3H-, purely 14C- and double (3H + 14C) labelling, is only possible if the activity ratio of 3H- to 14C-thymidine is sufficiently high. This condition is necessary for a reliable distinction between those grains in the first emulsion which are due to true 3H-labelling and spurious grains which are simultaneously produced in the same emulsion by 14C-beta-particles. Experiments are described to determine the required activity ratio of 3H- to 14C-thymidine.


Study of the effect of hydroxyurea on the erythropoietin-sensitive cells. The response of polycythaemic mice to a standard dose of erythropoietin has been measured at various time intervals after single or repeated injections of hydroxyurea. The results exclude S phase of the cell cycle as the period responsive to erythropoietin. They suggest the existence of feedback mechanisms within the cell cycle, operating at the G1--S boundary and within the G1 phase. Hydroxyurea given to polycythaemic mice at various time intervals after erythropoietin induced characteristic changes in the response. These changes can be explained if both gradual transit of differentiated cells into the DNA synthesis (S phase) and changes in amount of the erythropoietin sensitive cells caused by the feedback mechanisms operating in the cell cycle are considered.


Changes in liver cells ploidy of young rats following isoprenaline treatment. Cell proliferation induced by isoprenaline (IPR) stimulation in very high doses was assayed in the liver of young rats, and the formation of polyploid cells was studied form the 15th to the 70th day of life. A general stimulatory effect on a complex process of cellular multiplication, leading to a population of tetraploid cells, was found to be accelerated; the earlier appearance of binucleate cells and the subsequent significant variations in their incidence confirmed the role of this cell type as an intermediate step in the process of polyploidization. Evidence was found of concomitant size changes of the hepatocytes, which might be partially independent of the effect of DNA content. The stimulation was no longer evident 20--30 days from the end of treatment, by when the cells which had come into contact with IPR should have completed the whole sequence of events leading to the formation of tetraploid mononucleate cells.


Periodontal ligament cell kinetics following orthodontic tooth movement. The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (3H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with 3H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. 3H-TdR-labeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bbone forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G2 block by the orthodontic stimulation.


The extravascular channel system in the rostral pituitary of Mugil cephalus (Teleostei) as revealed by use of horseradish peroxidase. The interlobular spaces, separating the neuro- and the adenohypophysis can be visualized in the pituitary of the Grey Mullet by using the horseradish peroxidase technique. On the one hand, these spaces communicate with the pericapillary spaces. On the other, they are linked with the pericellular spaces in the adenohypophysis and the periaxonal spaces in the neurohypophysis, thus providing a pathway for the exchange of metabolites and hormones between blood system, epithelial cells and the terminal region of the neurosecretory axons. The interlobular spaces branch off between the tips of the corticotrop cells and form a network adjacent to the adeno- and neurohypophyseal interface. It is possible that branches of this network penetrate into the interior of the corticotrops. The probable role of the network is to permit close contact between neurosecretory hormones and the corticotrops and also to enable the corticotrop hormone to reach the capillaries.


Comparative ultrastructural investigations of the pineal organ of the blind cave fish, Anoptichthys jordani, and its ancestor, the eyed river fish, Astyanax mexicanus. A comparative ultrastructural study has been made of the pineal organ in specimens of two closely related populations of the characid fish, Astyanaz mexicanus. The specimens of one population are living in the river, under natural light conditions. The specimens of the other population, originally described as Anoptichthys jordani, are living in a completely dark cave. In specimens of both populations the pineal organ consists of a spindle shaped end-vesicle, connected to the diencephalic roof by a slender stalk. The pineal tissue is compact and consists predominantly of glia-like supporting cells and sensory cells resembling the photoreceptor cells of the lateral vertebrate eye. Phagocytotic microglia-like cells can be found in close contact with the outer segments of the sensory cells. Nerve cells are located in the neighbourhood of neuropil formations, in which synaptic contacts are established between sensory cells and nerve cells. From these nerve cells fibers are emerging, forming the pineal tract that runs down the pineal stalk towards the diencephalon. On the basis of the ultrastructure described by other authors it is concluded that the pineal organ in specimens of the river population of Astyanax mexicanus resembles the pineal organ of other fish species. In specimens of the river population, reared under normal light-dark conditions for 3, 9 or 18 months, conspicuous morphological changes have not been detected in the presumably light-sensitive outer segments of the sensory cells or in other parts of the pineal tissue. In specimens of the cave populations, reared under identical conditions, an age-dependent, gradual regression of the regular outer segment organization of the pineal sensory cells takes place. In other parts of the pineal tissue, only small morphological changes can be observed. In specimens of the cave population, reared in constant darkness, the regression of the pineal outer segment organization begins earlier and is obvious. It is postulated that the gradual age-dependent regression of the regular organization of the outer segments in the pineal organ of cave specimens of Astyanax mexicanus is genetically determined and indicates a regressive evolution of the pineal light sensitivity. The expression of the regressive traits is dependent on the environmental light conditions.


On the fine structure of the regressing ecdysial glands of carcinus maenas L. (Crustacea Decapoda) parasitized by Sacculina carcini Thompson. The ecdysial glands (Y organs) of the crab Carcinus maenas regress in the presence of an external parasite, Sacculina carcini. This regression is more or less severe and may lead to complete autolysis. Three gradual stages in this involutionary process are described. In stage I, the gland cells are nearly normal. Nuclei and cytoplasmic organelles remain unchanged, but large vacuoles begin to appear. Stage II corresponds to more or less drastic nuclear pyknosis and cytoplasmic alterations. Myelin figures are large and numerous. Lysosomes and autophagic vacuoles with phosphatase activity are abundant. However, the general cellular architecture remains preserved. Stage III corresponds to irreversible cytolysis; nuclear envelopes and plasma membranes have disappeared. What remains is an accumulation of cellular debris becoming engulfed by circulating hemocytes. Not all of the gland cells of any given Y organ show the same degree of regression; degeneration is asynchronous. Structures seemingly corresponding to absorptive roots of the parasite are seen. Their lumen is coated with microvilli. The putative direct and indirect influences of the rhizocephalan parasite on its host are discussed. Our results on regressing Y organs of parasitized crabs are compared with those on regressing ecdysial glands of insects.


The morphological categorization of developing synaptic junctions. The molecular layer of rat occipital cortex was studied at a number of ages throughout postnatal development in an attempt to a formulate a quantitative morphogenetic scheme of synaptic development. As previous work had pointed to the potential usefulness of E-PTA stained material in synaptogenic studies, this technique was employed. Synaptic junctions were assigned to five categories, A-E, on the basis of variations in the organization of their presynaptic densities. Of these categories, Type A represented a mature junctional form with well-defined and discrete dense projections. Type E, by contrast, represented the immature end of the spectrum, its presynaptic apposition generally lacking recognizable densities. Types B-D represented intermediate forms. As development proceeded, the percentage of A type junctions increased from 0.3% at 7 days to 25% in the adult. Type E junctions showed the opposite trend falling from 42% at 7 days to 5% in the adult. On the basis of these results, and those relating to types B-D, possible morphogenetic schemes involving the five synaptic categories are put forward. These schemes incorporate and extend those previously suggested by other workers. By placing observations on a quantitative footing, the schemes proposed here lend themselves to use in experimental investigations.


Arterio-venous anastomoses in rainbow trout gill filaments: a scanning and transmission electron microscopic study. The origin of arterio-venous anastomoses, connecting the efferent filament artery (EFA) with the central venous sinus (CVS) of gill filaments can be well discerned by scanning electron microscopy in the rainbow trout. Corresponding vessels between the afferent filament artery and the CVS could not be detected with the techniques applied. AVA-specific endothelial cells are characterized by their bulky shape and their microvillous surface. The general morphology of AVA's in Salmo gairdneri is very similar to that of AVA's in Tilapia mossambica (Vogel et al., 1974) but they are much longer in the trout. No filament whorls have been encountered in AVA endothelia of Salmo gairdneri.


Histological and ultrastructural studies of the rabbit pars intermedia in organ culture. II. Developing tissue. Pars intermedia (PI) tissue from fetal, perinatal, neonatal and juvenile rabbits has been maintained in organ culture for up to nine weeks after explantation. Autoradiography showed that DNA synthesis took place for at least 22 days of culturing. PI-glandular cells and interstitial cells remain identifiable throughout this period but ACT-type cells were recognised only up to six weeks. Material from fetal and perinatal animals had a higher proportion of surviving cells than that from adult animals. The degree of differentiation achieved by PI-glandular cells in vitro appears to depend on three factors: i) the stage of development reached before explantation; ii) the original topographic position in the PI tissue before explantation; and iii) the position in the explant in relation to the gas-liquid interphase.


Nucleolar DNA in oocytes of Salmo irideus (Gibbons). The amplification of ribosomal genes has been studied in oocytes from Salmo irideus. In situ nucleic acid hybridization showed that the synthesis of nucleolar DNA begins in oogonium and proceeds slowly through leptotene and zygotene when a small amount of extrachromosomal nucleolar DNA is produced. In early pachytene there is a rapid build-up of nucleolar DNA demonstrable by rapid incorporation of tritiated thymidine. Synthesis stops completely in early diplotene when nucleolar DNA becomes dispersed over the inner surface of the nuclear envelope in the form of small Feulgen-positive granules. Photometric measurements of Feulgen stained nuclei showed that the final amount of amplified nucleolar DNA synthesized in each oocyte is approximately 20 mug. The amplified DNA does not form a heterochromatic mass. The buoyant density of the amplified nucleolar DNA calculated from analytical centrifuge tracings in relation to DNA from Micrococcus luteus (rho = 1.731 g cm-3) is 1.715 g cm-3 and corresponds to a G+C content of 57%. There are indications that the buoyant density of the somatic nucleolar DNA is lower than that of amplified nucleolar DNA. Similarities and differences between ribosomal gene amplifications in oocytes of Salmo irideus and the corresponding process in Xenopus are discussed.


Surface morphology of lens fibers from eyes of normal and microphthalmic (Browman) rats. The surface features of cortical fibers from lenses of normal adult rats and microphthalmic rats of the Browman strain have been studied by scanning electron microscopy. In the normal lenses, superficial cortical fibers follow a straight course from inner to outer pole whereas the deeper cortical fibers, while straight near the poles, pursue an undulating or zig-zag course at and near the equator. Almost all of the fibers are hexagonal in cross section and all fibers throughout their entire length are bound by interdigitating processes at each corner of the hexagon to corners of two adjacent fibers. Some fibers are also affixed by a single row of ball and socket junctions located on their broad outer and inner surfaces. Lens fibers from Browman rats display both minor and major abnormalities. These included segmentation, formation of incisures and lateral protrusions, corrugation and villous-like alteration of the broad fiber surface and development of parallel ridges of broad surfaces in a basket-weave pattern.


Ultrastructure of the new neuromuscular junctions formed during reinnervation of rat soleus muscle by a "foreign" nerve. In rats the "fast" fibular nerve was transposed to the "slow" soleus muscle outside the original innervation band. Formation of new neuromuscular junctions was induced by cutting the soleus nerve after different periods of time. The morphological maturation of these junctions was studied by electron microscopy. New neuromuscular junctions do not form when the original innervation is left intact. Three to five days after denervation, vesicle-laden terminal boutons contact muscle fibers with only the basal lamina of the latter intervening. Three weeks after denervation, most boutons are larger and postsynaptic folds are present, although younger stages are also seen. Sixteen weeks after denervation, the neuromuscular junctions appear mature. This corresponds well with electrophysiological findings in the same material. The fully developed neuromuscular junctions sixteen weeks after denervation possess postsynaptic folds similar to those of normal "fast" muscle fibers. This suggests that the "fast" fibular nerve rather than the "slow" soleus muscle fibers determines the morphology of the postsynaptic folds. Possible trophic neuromuscular interactions are discussed.


Immunohistochemical localization of LH-producing cells in the adenohypophysis of the Japanese quail, Coturnix coturnix japonica. The cells that produce luteinizing hormone (LH) in the adenohypophysis of the Japanese quail were identified immunohistochemically using anti-chicken LH serum and horseradish peroxidase-labeled goat anti-rabbit gamma globulin serum. The LH cells are localized in the caudal lobe of the pars distalis. They are elongate in shape shape and are polarized toward the sinusoids, especially in their active states. Alterations in size of LH cells are directly related to changes in circulating LH levels as induced by castration or photostimulation. The LH cells identified immunohistochemically were only stained by alcian blue with periodic acid-Schiff (PAS), alcian blue and orange G. PAS-positive gonadotropic cells in the cephalic lobe were stained immunohistochemically only slightly if at all using anti-chicken LH serum and consequently may be FSH producing cells. In the cephalic lobe another type of basophilic cell was stained with alcian blue. These cells were also stained immunohistochemically with anti-chicken LH serum. These cells may possibly be identified as TSH cells due to the characteristics of the anti-chicken LH serum used in this study which cross react with LH and TSH but only slightly with FSH, and also on the basis of previous light and electron microscopic studies.


The effects of testosterone, 5alpha-dihydrotestosterone, 3alpha-androstanediol, and 3beta-androstanediol on epithelial fine structure of the rabbit epididymis in organ culture. The fine structure of the corpus epididymidis of the rabbit has been studied following organ culture. Various modifications of tissue preparation and culture conditions were examined to obtain good maintenance of cellular integrity as well as to preserve sperm fertilizing ability. After 5 to 7 days in culture in the absence of hormonal support, the epididymal epithelium showed signs indicative of cellular regression. Such changes included shrinkage of the cells, loss of the border of stereocilia, decrease in smooth endoplasmic reticulum, and an increase in autophagic vacuoles. The presence of androgens in culture media prevented cellular regression to varying degrees, depending on the hormone utilized. With regard to maintenance of cellular integrity, potency of the androgens tested was as follows: 5alpha-dihydrotestosterone greater than or equal to 3alpha-androstanediol greater than testosterone greater than 3beta-androstanediol. Addition of insulin to dihydrotestosterone-containing cultures resulted in no improvement in maintenance. Phagocytosis of spermatozoa by epithelial cells was observed in cultured tubules and the degree of spermiophagy was inversely proportional to successful maintenance of fine structural characteristics of epithelial cells. The morphological findings reported here correlate well with the fertilizing ability of spermatozoa from cultured epididymis as reported in an accompanying communication.


The effects of testosterone, 5alpha-dihydrotestosterone, 3alpha-androstanediol, and 3beta-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture. The fertilizing ability of spermatozoa from epididymal tubules maintained in organ cultures from 1 to 7 days was assessed after artificial insemination into receptive does. It was found that spermatozoa from the distal corpus which were already capable of fertilizing eggs prior to the cultures retain this ability for 1 day without addition of hormone and for 3-4 days when testosterone (0.5 mug/ml) or 5alpha-dihydrotestosterone (0.5 mug/ml) is added to the culture medium. Spermatozoa from the proximal corpus which were not capable of fertilizing eggs prior to the cultures remain so after 1 day in cultures without addition of hormone. Testosterone, 5alpha-dihydrotesterone, 3alpha-androstanediol, or 3beta-androstanediol was added to cultures of proximal corpus at a concentration of 0.5 mug/ml. Only with 5alpha-DHT is the mean percentage of fertilization significantly higher than the percentage obtained without addition of hormone. Insulin does not potentiate the effect of 5alpha-DHT on sperm fertilizing ability. Epithelial growth factor is ineffective. Spermatozoa from the caput epididymidis kept in cultures for 1 to 4 days remain infertile. The results are discussed in light of the morphological findings presented in the preceding communication and in relation to the physiological requirement for sperm maturation in the epididymis.


Studies on the innervation of the endometrium. The innervation of the endometrium of rabbit, rat, mink, mongoose and pig has been investigated electron microscopically. Large bundles of nerve fibers can be observed in the connective tissue spaces within the basal layer of the endometrium. Unmyelinated nerve fibers enter the lamina functionalis, terminal nerve fibers penetrate the basal lamina and make contact with the glandular and the cavum epithelial cells. The terminal axons contain abundant synaptic vesicles, dense core vesicles and mitochondria. To date, no specialized presynaptic or postsynaptic membranes have been found.


High voltage electron microscopy of the optic neuropile of the housefly, Musca domestica. Synaptic cartridges of the first optic neuropile (lamina ganglionaris) of the housefly were examined by high voltage electron microscopy (HVEM). Stereo pairs (from thick, i.e., 0.25 mum, sections viewed at 1,000 kV) provided a three dimensional representation of cartridge neurons and clearly revealed the lateral spread, bifurcation and some functional associations of Type I (L1, L2) monopolar interneurons. Slightly proximal to cartridge neck level, pairs of retinular (R) axons made contact with each other and it appeared that R processes projected through the cleft between the Type I interneurons. No junctional modifications were seen between contiguous R axon terminals. The speculation was made that functional contact might exist between neighboring R axons prior to their extensive synapses with principal first order interneurons. Such alleged coupling between R axons would account for several electrophysiological findings from other laboratories. Modifications in EM technique applicable for HVEM were detailed. The value of obtaining thick serial sections and the use of the HVEM in expediting three dimensional reconstructions of neuropile were demonstrated.


Retinohypothalamic projection in the mouse: electron microscopic and iontophoretic investigations of hypothalamic and optic centers. The problem of the direct retinohypothalamic projection in mammals (Moore, 1973) was reinvestigated in the laboratory mouse by electron microscopy and cobalt chloride-iontophoresis. The time-course of the axonal degeneration in the suprachiasmatic nucleus was studied 3, 6 and 12 h, 1, 2, 4, 6, 9 and 12 days after unilateral retinectomy. Specificity of the degenerative changes was controlled by investigation of the superficial layers of the superior colliculus. The ratio of crossed to uncrossed optic fibers could could be determined by counting degenerating structures (axons and terminals) in the optic chiasma and the ipsilateral and contralateral areas of the optic tract, the suprachiasmatic nucleus, and the superior colliculus. The number of degenerating axons in the suprachiasmatic nucleus showed a maximum one day after unilateral retinectomy and was, at all stages studied, two to three times higher in the contralateral than in the ipsilateral nuclear area. In the optic tract and in the superior colliculus the number of degenerating profiles was three times higher in the contralateral than in the ipsilateral area. Retinohypothalamic connections and crossing pattern of retinal fibers were studied light microscopically using impregnation with cobalt sulfide in whole mounts of brains. Most of the optic fibers in the laboratory mouse are crossed crossed (70-80%). A bundle of predominantly crossed optic fibers runs to the suprachiasmatic nucleus.


Microtubule--synaptic vesicle associations in cultured rat spinal cord neurons. This paper describes new ultrastructural features of neural processes and of synapses in cultured CNS tissue treated with albumin before fixation using a modification of the technique recently introduced by Gray (1975). Nerve fibre bundles in explants of foetal spinal cord grown in vitro for 15-18 days were transected microsurgically. After transection the cultures were exposed to 20% albumin in distilled water and then fixed in unbuffered osmium tetroxide followed by unbuffered glutaraldehyde. In this material, but not in controls (injured but not exposed to albumin; exposed to albumin without injury) microtubules were found within many axonal varicosities, often situated close to presynaptic membrane specializations. These microtubules were closely associated with vesicles resembling synaptic vesicles, which were occasionally aligned in rows along the microtubules. Similar vesicle-microtubule associations were also found in non-terminal axons. Microtubules were also observed very close to some postsynaptic densities. The possiblity that the microtubule-vesicle associations are involved in vesicle movements (along axons and/or within axon terminals) is discussed. A more direct involvement of microtubules in terminals in the mechanism of transmitter release is also considered.


Ultrastructural studies on the secretory process in the epithelioid cells of the juxtaglomerular apparatus. The epithelioid cells of the juxtaglomerular apparatus have been studied with respect to the release mechanism of the secretory granules. Invaginations of the plasma membrane into the interior of the epithelioid cells are interpreted as stages before or after an exocytotic process. Granules are sometimes observed in close contact with the plasma membrane, and material with electron density similar to that of the granules can also be observed in the invaginations. These morphological features suggest that the granular material of the epithelioid cells is extruded into the texture of the basal lamina. Furthermore, a dense network of microtubules and microfilaments is described and the functional role of this system in exocytosis is discussed.


Fine structure of chemosensitive cells (glomus caroticum) in tissue culture. Cells of the carotid body of both embryonic and 1 to 2-d-old rabbits were cultured in monolayer in primary tissue culture. The cells grew in their original association of type I and type II cells. The fine structure of both cell types was similar to that in vivo. Even after one week in culture their morphological characteristics, such as the dense-cored vesicles, were preserved. The prolonged synthesis of catecholamines in culture and the formation of new granular vesicles is discussed.


Export of protein from the osteoclast as studied by electron microscopic autoradiography. The present electron microscopic autoradiographic study includes a quantitative analysis of osteoclasts in vitro using tritiated leucin as a protein tracer. A significant increase in the grain density over the ruffled border and the underlying resportion zone was demonstrated two hours post pulse whereas the grain density of the remaining cytoplasm was relatively constant. This indicates a transport of newly synthesized protein from the osteoclast to the extracellular resorption zone. Earlier histochemical and biochemical experiments suggest that the exported protein may represent lysosomal enzymes to be used in the extracellular bone degradation.


Freeze-fracture study of the junctional complexes of human and rabbit thyroid follicles. Zonulae occludentes, gap junctions and desmosomes have been demonstrated in replicas of freeze-fractured follicular cells of normal human and rabbit thyroid glands. The zonulae occludentes between the human follicular cells are composed of two to eight strands, which completely separate the intercellular space from the follicular lumen. Four to twelve or more strands are visible between the follicular cells of the rabbit thyroid gland. In the meshes of the zonulae occludentes as well as below them, gap junctions are present. They are numerous on the fracture faces of the human follicular cell membranes, but infrequent in those of the rabbit. Aggregates of particles related to desomosomes are found in the deeper meshes of the zonulae occludentes or close to them.


Identification, in the external region of the rat median eminence, of separate neurophysin-vasopressin and neurophysin-oxytocin containing nerve fibres. Immuno-enzyme cytochemical investigations, using single and double staining techniques, showed that the external region of the rat median eminence contains separate neurophysin-vasopressin fibres and neurophysin-oxytocin fibres. These neurophysin-hormone containing nerve fibres are influenced by bilateral adrenalectomy and by colchicine treatment. The external region of the median eminence of the homozygous Brattleboro rat contains neurophysin-oxytocin fibres. It does not contain immuno-reactive neurophysin-vasopressin fibres. Bilateral adrenalectomy also influences the neurophysin-vasopressin containing neurons of the suprachiasmatic nuclei. In the neurons of the parvicellular part of the rat hypothalamic paraventricular nuclei, staining for vasopressin and for oxytocin is completely absent.


Immunohistochemical localization of prolactin cells of the pars distalis in the fetal and neonatal hamster: a light and electron microscopy study. Using the immunoperoxidase technique, a small number of prolactin cells were first detected in the pars distalis of the hamster near developing sinusoids at 13 1/2 days gestation. Little change in number or distribution of immunoreactive cells was noted until the first few days after birth when a dramatic increase in number of immunoreactive cells was demonstrated throughout the pars distalis. Electron microscopy revealed cells in the fetal and neonatal anterior pituitary which had immunoreactive granules smaller in diameter than those seen in adult pituitary cells.


Ultrastructure of the aortic diverticula of the adult dragonfly Sympetrum danae (Odonata: Anisoptera). The aorta of Sympetrum danae possesses two dorsal diverticula: one in the mesothorax and one in the metathorax. They are very similar in form and position. Each diverticulum has a dorsal valve through which blood is pumped from the wings down into the aorta. The wall of the aortic diverticula consists of two simple cell layers: an outer epidermis-like layer and an inner muscle layer. The nuclei of the muscle cells are situated close to the lumen of the diverticula. The mitochondria are evenly dispersed between the myofibrils and are often paired up on either side of the Z-band. The Z-bands are thick and fragmented. The length of the sarcomeres varies from 3.3 to 6.1 mu. The A-band length is about 3 mu. The myofibrils consist of thick (250 A) and thin (85 A) filaments. Each thick filament is surrounded by 9-12 thin filaments. The sarcoplasmic reticulum is well developed and separates the myofibrils with one or two layers. The T-tubules are flattened and branch irregularly like a two-dimensional tree between the lamellar myofibrils. Intercalated discs are observed. The peculiarities of the muscle of aortic diverticula in S. danae are discussed in relation to various muscles of other insects and arthropods.


The ocellus of the cockroach, Periplaneta americana (Blattariae): receptory area. The ocelli of Periplaneta americana were studied by light and electron microscopy. The view that the ocellus of the cockroach represents a degenerated structure can no longer be supported. All organelles necessary for function are present. The club-shaped retinular cells lie homogeneously distributed in the cupule of the ocellus. Rhabdoms are seen as sickle-, y-, x- or star-shaped structures with up to six cells in formation. Cells were found which had formed two rhabdomeres. The mass of cell organelles lies in the cytoplasm between the cell nucleus and the rhabdom. Smooth endoplasmic reticulum is wound into a spindle formation of considerable size at the origin of the axon in some cells. A cylindrical body in which 10-40 microtubules are packed, as yet unknown in insect retinular cells, is described. The receptory area of the ocellus terminates in a tapetum which contains granules, soluble in alcohol. The axon bundles of the retinular cells run through the tapetum and immediately thereafter make synaptic contact with dendrites of the ocellar nerve cells, while still within the ocellus.


Light and electron microscopic studies on the pars intermedia of the chameleon. The neurointermediate lobe of the hypophysis in the Chameleon (Chamaeleo dilepis) was examined with light and electron microscopic methods, with special reference to the cytology of the pars intermedia (PI). The PI is the largest lobe of the hypophysis consisting of (1) dark cells with secretory granules ranging from 200-600 nm; (2) light cells, far fewer in number, containing granules 150-300 nm in diameter; (3) stellate, non-secretory cells. The secretory cells abut onto the perivascular basal lamina of the capillary sinusoids while their apical part borders an intercellular space. This surface of the cells often bears a cilium. The granules arise from the Golgi cisternae while small detached vesicles are found between circumscribed sites of the cell membrane and the Golgi apparatus. No nervous elements were found in the pars intermedia and it is assumed that the regulation of this lobe is purely humoral. This is supported by the presence of three types of nerve terminals in the pars nervosa: (a) terminals with large secretory granules and small vesicles; (b) terminals with dense-core vesicles and small vesicles; (c) terminals with small vesicles only. All of these are secretory as indicated by the presence of the synaptic semidesmosomes formed with the perivascular basal lamina.


Ultrastructural features of adult human tendon. A variety of human tendons have been studied at the electron microscope level. The fibers of these tendons are composed of collagen fibrils that average 1,750 A and 600 A in diameter. A third population that measures 100 A in diameter may represent immature collagen or filaments that are incorporated into tendon elastic fibers. The larger collagen fibrils vary in ratio with respect to one another, and are connected by interfibrillar bridges which in some cases appear to extend through the substance of the fibril. The collagen fibrils of the paratenon are less-well organized than those of the tendon proper and average 600 A in diameter. Tendons that exhibit the property of lateral stretch (plantaris and palmaris) were compared at the ultrastructural level with tendons that do not have this property. No differences between the two tendon types could be determined in normal or spread preparations, indicating that the differences in physical characteristics are a result of fiber rather than fibril organization.


Electron microscopic study on the gel of the central part of the corpus vitreum in the ox. The central part of the corpus vitreum in the ox possesses a relative firmness. Electron microscopically it has been shown to consist of collagen fibrils with interfibrillar spaces containing 8 nm thick granules. The granules are made up of chains of macromolecules (hyaluronic acid in an oligomer state) 130-200 nm in length and of an oval shape. The collagen fibrils are tightly covered with 8.5 nm thick macromolecules which represent highly polymerized hyaluronic acid. These macromolecules can be stained with ruthenium-red.


Arapen: a secondary complex between platinum ethylenediamine dichloride and cytosine arabinoside. I. Preparations and reactivity towards nucleic acid constituents. An equimolecular complex between platinum ethylenediamine dichloride (PtenCl2) and 1-beta-D-arabinofuranosyl cytosine (araC) has been prepared (arapen). This 1 : 1 complex in which both ethylenediamine and araC moieties carried a radioactive label was found to be unreactive towards cytosine and guanine derivatives, but to react to the extent of 30% with deoxyadenosine, adenosine 5' -monophosphate, polyadenylic acid, poly a(AT) and DNA at comparable rates. It is suggested that adenine compounds react with platinum complexes such as PtenCl2 in a bidentate manner, but are still able to react with arapen as monodentate ligands. However, hydrolysis of the last chloride ion from arapen may not be complete, and the incoming adenine nucleophile is only able to react to a limited extent.


Transfer of methyl groups from N-dimethylnitrosamine to glycerolipids in rat liver. We have examined the in vivo labeling of lipids after a single intraperitoneal injection of the carcinogen, (C14) dimethylnitrosamine, into rats. Liver was most active in incorporating (C14) methyl groups into lipids (0.91% of the injected dose) and 80% of the activity appeared in sn-3-phosphatidyl-choline. Chromatographic analysis of the products (and derivatives) formed after treatment of the (C14) phosphatidylcholine with phospholipase A2 (EC 3.1.1.4) and phospholipase C (EC 3.1.4.3) demonstrated that 89% of the radioactivity was in the choline moiety. These results indicate the transfer of methyl groups to lipids occurred via the lipid methylation pathway that converts phosphatidylethanolamine to phosphatidylcholine.


The binding of dehydroheliotridine to DNA and the effect of it and other compounds on repair synthesis in main and satellite band DNA. This study was aimed at elucidating the mechanisms of the preferential depression of satellite DNA synthesis by dehydroheliotridine (DHH). DHH was found to induce repair synthesis to the same extent in both main and satellite band DNA in cultured sheep lymphocytes. This was also the case with acridine orange, nitrogen mustard (HN2) and ethyl methane-sulphonate (EMS). Using analytical equilibrium ultracentrifugation no difference was found between the extents of in vitro binding of DHH by main and satellite band DNA. From these results it was concluded that the depression of the synthesis of satellite DNA could not be explained by either its preferential binding of DHH or by less effective repair mechanisms. Radiolabelled DHH when added to synchronized cultures of ovine kidney cells was found to be preferentially bound to the satellite DNA (one DHH molecule to 6000 nucleotides) compared with the main band DNA (one to 10 000). When 5-bromodeoxyuridine (BUdR) was added to the cultures no DHH label was found in the heavy, semiconservatively replicated DNA band. From these findings it is suggested that attack may occur during mitosis where all of the satellite DNA may be undergoing synthesis at the same time, thus explaining the increased amount of DHH bound to the satellite.


Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse. The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.


DNA repair synthesis in guinea pig pancreatic slices following in vitro exposure to N-nitrosomethylurethane. The incorporation of thymidine into DNA in the presence of hydroxyurea (HU) by guinea pig pancreatic slices following exposure to N-nitrosomethylurethane (NMUT) was used to follow DNA repair synthesis. HU was used to suppress normal replicative DNA synthesis. Slices from the duodenal segment of the pancreas were exposed for periods of 15 to 90 min to NMUT at concentrations of 2 to 20 mM, then incubated in tritiated thymidine ([H3]-TdR) free of carcinogen, and radioactivity in DNA was determined. NMUT induced a a dose- and time-dependent increase in HU-insensitive thymidine incorporation. This stimulated incorporation, which could be attributed to repair synthesis, occurred immediately following the treatment and was largely complete within 3 h.


Studies on pulmonary aryl hydrocarbon hydroxylase activity in inbred strains of mice. Pulmonary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene (MCA). I.t. instillation of 188 mug MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/cMai, C3H/fMai, and C57L/J were observed to be induced within 24 h after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/2J and RF/J expressed no such increase. At a dose of 500 mug MCA, the pulmonary tissue of DBA/2 mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7,8-benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.


The role of liver glutathione in the acute toxicity of retrorsine to rats. (1) Two procedures have been used to change the glutathione concentration in the livers of male rats. The glutathione level is increased to about double that of the controls, 0.5 h after the administration of cysteine (200 mg/kg, i.p.) and to about 25% that of controls, 1 h after the administration of 2-chloroethanol (30 mg/kg, i.p.). (2) The acute LD50 of retrorsine to rats (42 mg/kg) is increased by pretreatment with cysteine to 83 mg/kg and decreased by pre-treatment with chloroethanol to 23 mg/kg. In all three groups, deaths are accompanied by haemorrhagic centrilobular necrosis of the liver. (3) 2 h after the administration of retrorsine to rats (60 mg/kg), the levels of pyrrolic metabolites in the livers of animals pre-dosed with cysteine or chloroethanol are respectively about 60% and 200% those of rats given no pre-treatment. (4) Neither in the normal nor in the pre-treated rats dose retrorsine (60 mg/kg) cause a detectable fall in liver glutathione concentration 0.5-4 h after dosing. By 24 h, the glutathione concentration in the livers of the retrorsine-dosed rats is higher than those of the corresponding controls. There was no significant change in the liver weights of the treated rats relative to the controls. (5) Treatment of rats with retrorsine (60 mg/kg) causes a fall in the liver concentrations of cytochrome P-450, 24 h after dosing. This loss of cytochrome P-450 is increased in rats pre-treated with chloroethanol. The concentrations of cytochrome b5 in the same animals are not significantly reduced.


The synthesis of chiral glycerides starting from D- and L-serine. A method for synthesizing chiral glycerides starting from L- or D-serine is described. Optically-active serine (both enantiomers are commerically available) was transformed into glyceric acid by stereospecific diazotization. The configuration at carbon atom 2 was maintained during the reaction. The glyceric acid was then converted into optically pure isopropylideneglycerol - which is an important intermediate in the synthesis of mono-, di- and triglyderides - by esterification followed by acetalization with acetone and reduction with lithium aluminium hydride. Reaction of this intermediate with triphenylphosphine in tetrachloromethane followed by acid-catalysed hydrolysis and dehydrohalogenation provided optically-active glycidol (2,3-epoxy-1-propanol). The epoxy ring of an ester of glycidol and a fatty acid was then opened stereospecifically with retention of configuration by heating the glycidol ester in the presence of a second fatty acid and a catalyst. This yielded a chiral 1,3-diglyceride which could be converted into a chiral triglyceride.


Gas chromatographic analysis of synthetic glycidol esters, mono-, di- and triglycerides. The gas chromatographic analysis of glycidol esters and mono-, di-,and triglycerides of palmitic-, stearic-, and oleic acid mixtures is described. The composition of the products was determined by gas chromatography on OV-17 after trimethylsilylation. Base-line separations between 1- and 2-monoglycerides and between 1,2- and 1,3-diglycerides were obtained. Isomerisation of the trimethylsilyl ethers of monoglycerides was not observed, contrary to published work.


Preparation and properties of vesicles enclosed by fatty acid membranes. Stable preparations of microscopic particles were obtained from long-chain fatty acids by mechanical agitation of evaporated films in presence of buffer solutions. Oleic and linoleic acids were used. Studies of osmotic swelling and shrinking of the particles indicated that they are enclosed by semipermeable membranes. The particles, which were named ufasomes, are also capable of entrapping glucose in spaces inaccessible to enzymes. It was concluded that the ufasomes closely resemble phospholipid liposomes in their structure and properties.


Vascular capacitance and fluid shifts in dogs during prolonged hemorrhagic hypotension. The mean circulatory pressure (Pmc) in dogs anesthetized with chloralose-urethane was estimated from 0.5 to 150 minutes after hemorrhages of 0, 17, or 34 ml/kg, or that volume giving an arterial pressure (Pa) of 40 mm Hg. The Pmc was determined by fibrillating the heart and then rapidly pumping blood from aorta to vena cava until Pa=venous pressure (Pv)=Pmc. Within about 10 seconds, the heart was defibrillated. Vascular compliance was estimated as the ratio of a test blood volume change (0, +/- 8.5, or 17 ml/kg) to the change in Pmc, determined 0.5 minute after the start of the test volume change. Erythrocyte and plasma volumes were measured by 51Cr-erythrocyte and 125I-albumin dilution. In response to prolonged hemorrhage: (1) the total vascular compliance apparently decreased; (2) most of the changes in capacity vessels occurred within 5 minutes, and indeed a large part of the response probably had occurred by the time of the first measurement at 30 seconds; (3) the progressive recovery in Pmc after 5 minutes was primarily from fluid shifting into the vasculature; (4) even after 2 hours of severe hemorrhagic hypotension, the venoconstriction was not lost; but (5) after an hour of severe hemorrhagic hypotension (arterial pressure of 40 mm Hg) there was water loss from the vasculature, because plasma protein and erythrocyte concentrations and plasma oncotic pressure increased.


Atrial receptors with nonmedullated vagal afferents in the cat. Discharge frequency and pattern in relation to atrial pressure. The response of 14 atrial receptors with nonmedullated vagal afferents to changes in atrial pressure was determined in anesthetized thoracotomized cats. Recordings were made from 11 single fibers and three two-fiber filaments. Mean conduction velocity was 0.9 m/sec (range, 0.35 to 2.2 m/sec). During the control period, either no activity was detected or there was a sparse discharge (mean, 1.4 impulses/sec), which occasionally was related to the a or v waves. As atrial pressure was increased by transfusion or by occlusion of the aortic, pulmonary arterial, mitral, and tricuspid orifices, an increased rate of firing occurred, often related to the atrial v wave. The threshold for individual receptors was between 2 and 3 mm Hg (mean pressure) in the right atrium, and 5 and 12 mm Hg in the left atrium. The maximal firing rate was 5-11 impulses/sec for right atrial and 10-20 impulses/sec for left atrial receptors. The receptors were localized by probing the opened heart, and were identified in both atria, in the interatrial septum, and in the atrial-venous junctions. Thus receptors connected to vagal C fibers are present throughout both atria and are activated by moderate changes in pressure.


Electrogenesis of increased norepinephrine sensitivity of arterial vascular muscle in hypertension. The possibility that the vascular muscle cell might contribute to the development of essential hypertension by being more responsive to norepinephrine because of an inherently lower membrane potential (Em) was investigated. Experiments were designed to test the hypothesis that Em of arterial vascular muscle cells from spontaneously hypertensive rats (SHR) are less negative than those from matched Kyoto normotensive rats (KNR). The caudal artery, a muscular, densely innervated regulating artery 300-400 mum in outside diameter, which is activated by graded (nonspiking) depolarization to produce a maintained contraction, was studied. Vascular muscle cells from SHR always had less negative Em than those from KNR at 16 degrees C, but not at 36 degrees C, over a range of K+ concentrations from 2.7 mM to 150 mM. From the relationship between Em and K+ concentration, intracellular K+ concentration ([K+]i) was estimated to be 150 mM for SHR and 170 mM for KNR. The caudal artery undergoes a large depolarization when K+ is removed from the superfusing solution and a transient hyperpolarization that exceeds the calculated EK (potassium equilibrium potential) when K+ is replaced. The magnitude of the hyper-polarization on returning to 30 mM or 50 mM K+ always was greater for vascular muscle of SHR than KNR. The apparently lower [K+]i and more active (compensating) electrogenic ion transport in the SHR vascular muscle cells thus result in an unaltered Em at body temperature in the physiological range of K+ concentrations. However, depolarization by norepinephrine was greater over the middle of the dose-response curve, and this greater depolarization caused the contractions of SHR arteries to be greater. The altered electrogenesis of the SHR vascular muscle cells is postulated to provide a mechanism for the increased reactivity of arteries to norepinephrein in hypertension.


The relationship between overdrive suppression and overdrive excitation in ventricular pacemakers in dogs. We studied the excitatory and inhibitory effects of overdrive on idioventricular pacemakers in anesthetized dogs with recently induced complete atrioventricular block. The following results were obtained: (1) a slow driving rate may induce a temporary rhythm which may be reinstituted with additional stimuli; (2) the induced rhythm may appear as coupled extrasystoles which, on interruption of the drive, are found to be self-sustaining; (3) during continued slow driving, extrasystoles may appear and disappear in a cyclical manner; (4) a short period of fast driving may be followed by a fast new rhythm, the rate and duration of which are a function of the rate and duration of drive; (5) fast driving may induce a new rhythm at a rate below predrive control; (6) after a long period fast driving, only suppression follows; and (7) intermittent periods of fast driving lead to a summation of inhibition with each successive period. These results suggest the following conclusions: (1) under certain conditions, electrical driving instead of inducing suppression may induce a rhythm ("overdrive excitation") at a rate similar to, faster then, or slower ("inhibited excitation") than control; (2) the duration of diastole and the number of driven beats are major factors in the induction of new rhythms; and (3) overdrive excitation is counteracted by overdrive inhibition, with development of the former requiring fewer beats than the latter.


Abnormal ion and water composition of veins and normotensive arteries in coarctation hypertension in rats. We examined the water, sodium, and potassium composition of the thoracic aorta, abdominal aorta (plus iliac arteries), and veins (vena cava and portal vein) from rats with aortic coarctation. The aortas of 10 rats (group A) were coarcted above the renal arteries to produce hypertension. Control groups consisted of 10 rats sham-coarcted above and 10 rats coarcted below the renal arteries. In group A rats heart weights and carotid artery pressures were elevated over controls (P less than 0.01), whereas there were no significant differences in femoral arterial pressures. In group A rats both the hypertensive thoracic aorta and the normotensive abdominal aorta contained about 20% more water per unit of wet weight, and about 35% and 60% more sodium and potassium, respectively, per unit of dry weight than did the corresponding portions of aorta from control rats (P less than 0.01). In group A rats water (P less than 0.01), sodium (P less than 0.02), and potassium (P less than 0.05) contents of veins also were increased. There were no significant correlations between level of carotid arterial pressure and magnitude of changes in arterial and venous composition, nor were there significant differences between the magnitude of changes in the normotensive and hypertensive portions of the aorta. These results indicate that in rats abnormalities in vascular wall salt and water content are not necessarily a direct effect of the elevated pressure in hypertension.


Role of resistance and exchange vessels in local microvascular control of skeletal muscle oxygenation in the dog. The effects of reduction in perfusion pressure, arterial hypoxia, muscle contraction, and adrenergic stimulation on the hindlimb muscle circulation were studied. Under normal conditions (venous PO2 greater than or equal to 40 mm Hg), oxygen delivery to the muscle was maintained mainly by large increases in the capillary exchange capacity and the oxygen extraction ratio in accord with tissue demand following the application of the above stresses. The participation of the resistance vessels under these conditions was minimal. The prevailing venous oxygen tension then was reduced by several means and the response of vascular resistance and capillary exchange capacity to the same stresses was reexamined. At the lower prevailing venous PO2, the sensitivity of the resistance vessels to metabolic and hemodynamic disturbances was greatly increased. Consequently, blood flow autoregulation, functional hyperemia, and hypoxic hyperemia were more intense when venous oxygen tension was low. In contrast, the contribution of exchange capacity was diminished, probably owing to the fact that most of the capillaries already are open at low venous PO2. These data suggest that the locus of local microvascular control of muscle oxygenation shifts from the normally more sensitive precapillary sphincters to the proximal flow-controlling arterioles as the prevailing venous oxygen tension falls. Yet, although the relative contribution of the resistance and exchange vessels to intrinsic regulation of tissue oxygenation is related to the prevailing venous oxygen tension, the two compensatory mechanisms operating in concert maintain tissue PO2 above the critical level over a wide range of stresses.


Experimental evidence for regional cardiac influence in body surface isopotential maps of dogs. Isopotential mpas based on 192-200 body surface electrocardiograms were obtained for 20 dogs during multiple patterns of ventricular activation. The purposes of the study were to determine whether the cardiac location of events responsible for surface potentials had a recognizable influence on surface potential patterns and to examine the influence of electrical events occurring simultaneously in multiple cardiac regions. Substantially different effects of electrical activity in various cardiac regions on body surface potentials were evidenced by the body surface location of potential maxima and minima and by patterns of isopotential lines during early portions of ventricular excitation initiated at different ventricular sites. Simultaneous stimulation at some sites gave surface potential distributions with multiple extrema. These were demonstrated to be due to effects of the different cardiac regions, because addition of potentials due to stimulation of the individual sites duplicated those associated with simultaneous stimulation of the same sites. It was also shown that body surface locations of maxima and minima are not related in the same manner to the cardiac location of the responsible events when these events are present in single and multiple regions. Slopes of potentials due to events in single cardiac regions were shown to combine with slopes produced by events in other regions to yield maxima or minima at new body surface locations. Results of the study support the possibility of regional cardiac examination by electrocardiography but suggest that this will require quantitative descriptions of the details of potential patterns in addition to the location of potential peaks.


Maintained stroke volume but impaired arterial oxygenation in man at high altitude with supplemental CO2. Hypobaric hypoxia causes hypocapina and alkalosis, hemoconcentration and increased hematocrit, and a decreased cardiac stroke volume. To assess the role of the hypocapnic alkalosis in causing these other changes, five men were exposed to hypobaric hypoxia at a barometric pressure (PB) of 440 torr with an alveolar O2 tension of 55 torr for 5 days with 3.77% CO2 added to the atmosphere to prevent alkalosis. They did not lose weight, and arterial CO2 tension, pH, and cardiac stroke volume were unchanged. An unchanged hematocrit implied an unchanged plasma volume. During exercise to maximum, stroke volumes equaled sea level values but arterial hypoxemia was profound, the arterial O2 tension being 39 torr. By contrast, three men at high altitude without CO2 supplementation (PB=455 torr; alveolar PO2=56 torr) had weight loss, hypocapnia, alkalosis, and decreased stroke volume. Increased hematocrits suggested decreased plasma volumes. During exercise, arterial PO2 (48 torr) was higher than in the group receiving CO2. Maximum oxygen uptakes were decreased to a similar degree in the two groups. Catecholamine excretion doubled in the group with CO2 but in the group without CO2 catechoamine excretion was unchanged. A normal pH at high altitude apparently maintained plasma volume, which, with the increased  catecholamine excretion, may have prevented a decrease in stroke volume. However, the subjects with CO2 added did not have enhanced oxygen transport, because their arterial oxygenation was impaired.


Mechanical stimuli exciting type A atrial vagal receptors in the cat. The activity of type A right atrial vagal receptors was recorded from the right cervical vagus in cats anesthetized with sodium pentobarbital, immobilized with gallamine, and with their chests open. Nerve impulses initiated by receptor activation were recorded simultaneously with instantaneous right atrial pressure and dimensional changes under various hemodynamic conditions. Atrial volume changes induced by infusion of saline, bleeding, and occlusion of the inferior vena cava did not alter consistently the systolic activity of the receptors. Electrical stimulation of the right stellate ganglion significantly increased the frequency of discharge during systole, whereas electrical stimulation of the left thoracic vagus significantly reduced the frequency of discharge. These inotropic interventions produced similar effects when the heart was paced at a fixed rate. Pacing the right atrial appendage increased the systolic discharge of the receptors only when at high rates the atrium contracted against closed atrioventricular valves. To investigate the influence of tonic efferent sympathetic activity on spontaneous receptor discharge, three receptors were studied before and after bilateral surgical stellectomy, and in cats with their chest closed three receptors were studied before and after infusion of propranolol. Both of these interventions markedly reduced the systolic activity. In addition to having effects on systolic activity, injection of saline, vagal stimulation, and sympathetic "denervation" always activated the receptors during filling. Our results indicate that: (1) the systolic discharge of type A receptors is a function of the active tension developed by atrial muscle during contraction; and (2) the pattern of discharge of the receptors during the atrial cycle depends on both the degree of atrial distention and the state and extent of contraction.


Substructure of intercellular junctions in freeze-fractured alveolar-capillary membranes of mouse lung. The purpose of this study was to examine by freeze-fracture the ultrastructure of intercellular junctions between mouse pulmonary endothelial and epithelial cells, and to relate this fine structure to that deduced from previous physiological and ultrastructural studies using tracer techniques. Junctions between capillary endothelial cells consist of one to three interconnected rows of particles which show occasional discontinuities. Small gap junctions are associated with these rows of particles in the arteriolar end of the capillary bed. At the venular end, the junctions consist of low profile ridges on the protoplasmic fracture (PF) face or complimentary grooves on the exoplasmic fracture (EF) face some of which have a sparse number of associated particles. The vascular junctions are similar to those of vessels in rat omentum and mesentery, and resemble "leaky" junctions described in renal proximal convoluted tubular cells. Tight junctions (zonulae occludentes) between type I  pneumocytes or between types I and II pneumocytes consist of a band of interconnecting ridges on the PF face and complimentary interconnecting grooves on the EF face. These continuous epithelial junctions have a structure that is typical of tight occluding junctions. Occasionally zonulae occludentes between type I and type II pneumocytes are discontinuous; this may be the result of cell translocation as pneumocytes are shed into the alveolus. Intravascular perfusion fixation at high pressure (140 cm H2O) had no discernible effect on the structure of endothelial or epithelial junctions. The appearance in our study of freeze-fractured pulmonary endothelial and epithelial junctions reveals clearly the physical basis for the results of ultrastructural tracer and physiological studies which have suggested that it is the alveolar epithelium rather than the endothelium that is the chief permeability barrier to small, water-soluble molecules.


Altered venous function in hypertensive rats. The vascular beds of the upper or lower body of rats were perfused through the aorta with oxygenated Krebs-Ringer solution containing dextran (7 g/100 ml), at 37 degrees C. Perfusion was stopped every 10 minutes, and the pressure rise in the jugular or the femoral vein was recorded during rapid infusion (15.3-90.0 ml/min) of Krebs-Ringer solution into the inferior vena cava. The following groups of rats were studied: (1) six male genetically hypertensive rats (GHR), 9-11 months old, New Zealand strain; (2) seven female GHR, 5-6 months old, New Zealand strain; (3) eight male spontaneously hypertensive rats (SHR), 4 months old, Okamoto strain; (4) five male rats with two-kidney Goldblatt hypertension (2-KGH), 30 days postclipping; (5) seven male 2-KGH rats, 65 days postclipping; (6) eight male rats with one-kidney Goldblatt hypertension (1-KGH), 40 days postclipping; (7) weight- and sex-matched normotensive control rats of the appropriate strain; and (8) weight- and sex-matched two-kidney and one-kidney, sham-clipped normotensive rats. Preliminary studies showed that rapid infusion into the venous circulation fills the veins, but there is no entry of fluid into the arterial side of the circulation. Compared to controls, the venous pressure-volume curves of GHR (male and female), SHR, 1-KGH rats and 2-KGH rats, 65 (but not 30) days postclipping, were shifted toward the pressure axis (P less than 0.05). The shift of the venous pressure-volume curves persisted following the reduction of vasoconstrictor tone by killing the rats or by the administration of sodium nitroprusside (0.1 mg/ml perfusate), or by both. The findings suggest decreased venous capacity in hypertensive rats.