THE OCCURRENCE AND DISTRIBUTION OF ATROPINESTERASE,AND THE SPECIFICITY OF TROPINESTERASES

Atropinesterase was found to exist in approximately one out of every four rabbits, and no relation could be observed between the incidence of the enzyme and season, sex, color, age, or weight. The occurrence of the enzyme was also shown to be unrelated to that of cholinesterase. The distribution of atropinesterase in the blood and organs of rabbits was studied; the animals devoid of the enzyme in their blood contained no demonstrable activity in any of the organ extracts tested. The presence of atropinesterase in frog liver, and its absence from the serum, has been confirmed. Hydrolysis of homatropine, but not atropine, by guinea pig liver was observed, while the serum was without action on either of the compounds. On this basis the possibility arises that guinea pig liver contains a homatropinesterase enzyme separate from atropinesterase. It was shown that lack of atropinesterase activity in certain rabbits is not likely to be due to the presence of a naturally occurring inhibitor. It has been demonstrated that contrary to previous indications neither fresh egg white nor yolk possess atropinesterase activity. The specificity of tropinesterases was investigated and evidence was presented for the possible existence of two distinct enzymes, cocainesterase and tropacocainesterase.     

THE ENCEPHALITOGENIC ACTIVITY AND MYELIN BASIC PROTEIN CONTENT OF ISOLATED OLIGODENDROGLIA

Abstract— Oligodendroglia isolated from frozen human brain induced EAE in the guinea pig when injected with Freund's complete adjuvant. An acid extract of the isolated cells was highly encephalitogenic. The disease was clinically and histologically similar to EAE induced with CNS white matter or myelin basic protein(MBP). The isolated cells were essentially myelin-free but contained a large amount of myelin basic protein as determined by polyacrylamide gel electrophoresis. It was shown that the cells can absorb ¹²⁵I-MBP during isolation and the possibility that contamination from extra cellular MBP is responsible for the encephalitogenic activity is considered.     

Clathrin is axonally transported as part of slow component b: the microfilament complex

During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal elements travel in either of two slow components, SCa (with tubulin and neurofilament protein) and SCb (with actin). Clathrin, the highly ordered, structural coat protein of coated vesicles, has recently been shown to be able to interact in vitro with cytoskeletal proteins in addition to membranes. The present study examines whether clathrin travels preferentially with the membrane elements or the cytoskeletal elements when it is axonally transported. Guinea pig visual system was labeled with tritiated amino acids. Radioactive SDS-polyacrylamide gel electrophoresis profiles from the major components of transport were coelectrophoresed with clathrin. Only SCb had a band comigrating with clathrin. In addition, radioactive clathrin was purified from guinea pig brain containing only radioactive SCb polypeptides. Kinetic analysis of the putative clathrin band in SCb revealed that it travels entirely within the SCb wave. Thus we conclude that clathrin travels preferentially with the cytoskeletal proteins making up SCb, rather than with the membranes and membrane-associated proteins in the fast component.     

THE OCCURRENCE OF TWO MYELIN BASIC PROTEINS IN THE CENTRAL NERVOUS SYSTEM OF RODENTS IN THE SUBORDERS MYOMORPHA AND SCIUROMORPHA

Extracts containing myelin basic proteins have been prepared from CNS tissue of representatives of the three suborders of Rodentia—Myomorpha, Hystricomorpha and Sciuro-morpha. Analyses of the extracts by electrophoresis at low pH showed that one type (L) of myelin basic protein is present in the CNS of all of the rodents examined (rat, mouse, hamster, guinea pig, chinchilla, prairie dog, woodchuck and squirrel). This protein is comparable in molecular size and charge to the CNS myelin basic proteins found in several other mammalian orders. In the CNS of the myomorphs (rat, mouse, hamster) and sciuro-morphs (prairie dog, woodchuck, squirrel) there is an additional type (S) of myelin basic protein of higher cathodic mobility and smaller molecular size. This additional protein is absent from the CNS of the hystricomorphs (guinea pig, chinchilla). These findings indicate that the presence of two myelin basic proteins originally reported in the CNS of the inbred rat is not an anomaly of inbreeding. These data further suggest that the presence of a single L-type CNS myelin basic protein might be a general characteristic of hystricomorphs, while the presence of both L- and S-type CNS myelin basic proteins might be a general characteristic of the myomorphs and sciuromorphs. Analyses by electrophoresis at low pH failed to reveal differences in mobility among either the L-type or the S-type CNS myelin basic proteins of the different species. In contrast, when electrophoresis was carried out cathodically at high pH, species-related differences in mobility were observed among the L- and S-type CNS myelin basic proteins.     

豚鼠卵巢囊淋巴孔的发现及卵巢囊超微结构研究

本实验通过扫描电镜等方法研究豚鼠卵巢囊及卵巢囊淋巴孔的结构,并探讨了卵巢囊及其淋巴孔的种属差异.实验结果首次报道豚鼠卵巢囊内、外层上皮均存在淋巴孔;豚鼠卵巢囊结构在输卵管走行、囊闭合程度及囊表面超微结构等方面与金仓鼠存在差异.结果提示豚鼠卵巢囊内、外层淋巴孔是沟通卵巢囊腔、卵巢囊淋巴系及腹膜腔的形态基础,可能是三者间物质转运的重要途径.卵巢囊淋巴孔可能影响物种的繁殖并参与囊腔内局部免疫反应.卵巢囊结构和发育程度与对应的输卵管伞端等结构及其他生殖特点相适应,研究结果丰富了生殖形态学和比较解剖学资料.     

肿瘤抑制基因APCmRNA在豚鼠脑中的分布

APC基因是1991年被发现的一类肿瘤抑制基因,它被定位于人第5号染色体5q21处。APC基因如发生缺失或突变,则易患直肠肿瘤,并伴有部分先天痴呆的病例。本工作在孟帆已获得的APC基因在豚鼠中的同源cDNA的基础上,完成了对它的亚克隆,并利用原位杂交和RNA酶保护分析的方法,对它在脑中的分布进行了研究。发现APCmRNA主要在海马、大脑和小脑中表达;嗅球中杂交信号稍弱,脑干中最弱。海马中阳性细胞主要是锥体细胞,小脑中则主要是内层颗粒细胞。在一个月大的豚鼠胚胎的脑中也观察到相似的表达型式。进一步的研究有助于我们更好地了解神经发育和先天痴呆发生的分子机制。     

Immunological relationships of an endogenous guinea pig retrovirus with prototype mammalian type B and type D retroviruses.

A retrovirus endogenous to guinea pig cells was earlier shown to be morphologically similar to type B and type D prototype retroviruses. Molecular hybridization techniques were used to show that guinea pig virus nucleotide sequences are endogenous to both domestic (Cavia porcellus) and indigenous (Cavia aperea) guinea pigs, but cannot be detected in the DNA of either other hystricomorph rodents or other mammals tested. Using radioimmunological techniques designed to detect interspecies relationships, the major internal polypeptide of guinea pig virus (p26) was shown to share three different sets of interspecies antigenic determinants with squirrel monkey retrovirus, viper retrovirus, and mouse mammary tumor virus. Thus, guinea pig virus appears to provide an evolutionary link between type B and D retroviruses.     

Experimental allergic aspermatogenic orchitis. 1. Isolation of a spermatozoal protein (AP1) which induces allergic aspermatogenic orchitis.

A unique highly soluble aspermatogenic protein (AP1) was isolated from guinea pig testes and was shown by immunofluorescence to occupy the outer surface of the sperm acrosome. This protein is a potent inducer of allergic orchitis and aspermatogenesis; as little as 0.2 mug induced orchitis in 60 percent of guinea pig tested. The AP1 protein, relatively small and neutral, is stable under acid conditions, but at pH 8.6 shows a variety of forms due either to aggregation or polymorphism. The purified AP1 protein appeared homogeneous by polyacrylamide gel electrophoresis at pH 2.7 and in sodium dodecyl sulfate and by immunoelectrophoresis using rabbit antisera to either the purified protein or the testes extract. It also showed a single band on immunodiffusion over a wide concentration range. The purification procedure consisted of delipidation with chloroform/methanol (2/1); acid extraction at pH 3.0; precipitation with 85 percent saturated ammonium sulfate; trichloroacetic acid extraction and gel filtration on Bio-Gel A-1.5; gel filtration on Bio-Gel P-10; chromatography on CM52 cellulose; and preparative gel electrophoresis at pH 2.7. Approximately 20 mg of purified AP1 protein were obtained from 5000 g of wet guinea pig testes. The AP1 protein induced an autoimmune disease characterized by infiltration of mononuclear cells around and within the seminiferous tubules (orchitis), followed by extensive damage and destruction of the germinal cells (aspermatogenesis). The course of the disease induced by this protein (0.5 to 1 mug) was essentially identical with that seen with whole testicular tissue or other purified fractions.     

Immunocytochemical study of the distribution of S-100 protein in the parathyroid gland of rats and guinea pigs

The distribution of S-100 protein in the parathyroid cells of normal and hypercalcaemic rats and guinea pigs was investigated. Previous studies had shown that the applied antibodies detect only the beta subunit of S-100 protein. S-100 protein was found in all parathyroid cells of rats aged between 1 and 720 days. In adult guinea pigs, S-100 protein was detectable in only a small proportion of parathyroid cells. The level of S-100 protein in individual cells exhibited considerable variation, particularly in guinea pig. Hypercalcaemia did not affect the distribution of S-100 protein in the parathyroid cells of either rats or guinea pigs. In both species, the presence of small groups of parathyroid cells in the central fragments of thyroid lobes was often noted.     

THE EXCHANGE OF PHOSPHATIDYLCHOLINE AND OF PHOSPHATIDYLINOSITOL IN SHEEP BRAIN

—The exchange of phospholipids between liposomes and brain mitochondria has been studied in the presence of pH 5·1 supernatant fluids derived from rat, guinea pig, sheep and ox brains. The exchange phenomenon was similar to that observed in liver and heart, but phosphatidylinositol and not phosphatidylcholine was the most rapidly exchanging phospholipid. The phosphatidylcholine exchange activity was purified 186-fold from sheep brain and the protein fraction contained two major and several minor protein species. The phosphatidylcholine and phosphatidylinositol exchange activities have been shown to have very similar molecular weights and isoelectric points. However, their behaviour in response to changes in liposomal surface charge suggested that separate proteins might be involved in stimulating the exchange of the two phospholipid classes.     

Affinity labeling of residues within Hv2 of guinea pig anti-azobenzenearsonate antibodies of different isotypes and from different strains

Anti-p-azobenzenearsonate (ARS) antibodies of IgG1 and IgG2 isotypes produced in inbred strain 13 and strain 2 guinea pigs were affinity labeled with N-(bromoacetyl)-3-[(p-arsonophenyl)azo]-L-tyrosine (BAAT) or N-(bromoacetyl)-p-arsanilic acid (BAA). BAAT was shown to modify approximately 50% of the binding sites specifically and BAA approximately 30%. Both reagents preferentially modified residues in the heavy (H) chain to the extent that it contained over 80% of the affinity label associated with the native molecule. At least 80% of label borne by the variable domain of the H chain (VH) was found in the second hypervariable region (Hv2). BAAT labeled all anti-ARS antibodies exclusively at position N-59, which contains a lysyl residue. BAA labeled predominantly tyrosine at N-57 and, to a lesser extent, lysine-59 and tyrosine-50. Comparison of Hv2 sequences in anti-ARS and in antibodies reactive with other haptens has shown that tyrosine at N-50 and N-57 as well as lysine at N-59 is distinctive of antibodies with anti-ARS specificity, thus implying their involvement in antigen binding. The predominant sequence of Hv2 was identical in anti-ARS IgG1 and IgG2 molecules induced in either inbred guinea pig strain following either carrier priming or conventional immunization. Although limited variability does occur among the various populations of anti-ARS antibodies in certain residue positions in Hv2, no significant differences in the binding affinities or in the indexes of heterogeneity were seen among the various kinds of anti-ARS antibodies.     

Erythrocyte membranes - compression of lipid phases by increased cholesterol content

Lipid and protein interactions were studied in guinea pig erythrocytes containing a normal or a two-fold increased amount of cholesterol. The electron spin resonance (ESR) spectra of cholesterol-loaded cells labeled with fatty-acid probes showed an increase in the local viscosity of the membrane as compared with control cells. This increase reflects changes in the interior of the lipid matrix of the membrane because the probes resisted destruction by ascorbate, were unaffected by the action of pronase, and gave spectra similar to those of liposomes. No differences were observed between control and cholesterol-loaded cells in the conformation of the membrane proteins by either the infrared spectra or the ESR spectra of cells labeled with maleimide probes.     

Fucoidin inhibits attachment of guinea pig spermatozoa to the zona pellucida through binding to the inner acrosomal membrane and equatorial domains

Our previous study has shown that fucoidin, an algal heteropolysaccharide, is a potent inhibitor of sperm-zona binding in the guinea pig, hamster and human. To visualize the surface site of fucoidin binding, a biotinated derivative (B-Fuc) of the native fucoidin was prepared. B-Fuc retained the inhibitory activity and was used in conjunction with FITC-avidin to localize its binding sites on guinea pig spermatozoa using fluorescence microscopy. In living acrosome-reacted spermatozoa, B-Fuc bound predominantly to the inner acrosomal membrane and equatorial segment domains. The binding was effectively competed by a 10-fold excess of native fucoidin, but not by a 10-fold excess of heparin or a 20-fold excess of biotinated normal rabbit serum IgG. B-Fuc binding patterns on dead spermatozoa were quite different from that of living spermatozoa. The post-acrosomal region, rather than the inner acrosomal membrane and equatorial domains, was intensely labeled. This indicates the importance of using living cells in assessing true surface binding sites whenever possible. We conclude that the inner acrosomal membrane and/or equatorial domains are critical for zona binding in the guinea pig.     

DISTRIBUTION AND OPTICAL ACTIVITY OF THE BASIC PROTEIN IN BOVINE PERIPHERAL NERVE MYELIN

Abstract— Antiserum to BF protein isolated from bovine spinal roots has been used to study the distribution of the protein in other species and tissues.
Significant amounts of protein could be demonstrated in bovine, pig and rabbit peripheral nerve myelin. It was, however, scarcely detectable in guinea pig peripheral nerve myelin. There was BF protein in rabbit spinal cord as well as in peripheral nerve, but little or no BF protein in the liver, kidney, muscle or brain. BF protein in bovine spinal cord was localized in the myelin. The ratio of the BF protein to the encephalitogenic protein in the spinal cord myelin was around 0.15:1.0. BF protein was extractable from peripheral nerve myelin by saline as well as by acid solutions.
The circular dichroism spectrum of the BF protein in aqueous solution suggested that this protein contained a very large amount of β-structure. This structure was not considered to be the result of acid denaturation because the protein purified from the saline extract of peripheral nerve also showed a similar spectrum.     

Evidence for Specific Polypeptide Chain Folding in Myelin Basic Protein from Reactions Between Fragments of the Protein and Monoclonal Antibodies

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)     

A CRYSTALLOGRAPHIC STUDY OF PURE CARBONMON-OXIDE HEMOGLOBIN

1. Photomicrographs of crystals of pure carbonmonoxide hemoglobin of the following species are presented; ox, sheep, hog, dog, turkey, rat, horse, chicken and guinea pig. Photomicrographs of the oxyhemoglobin crystals of the following species are also shown: ox, sheep, hog, dog, rat, horse and guinea pig. The crystals were formed from the pure protein by adding a suitable amount of ethyl alcohol and maintaining a temperature of 0°C., or lower. 2. In some species a sufficient difference is shown between the carbonmonoxide hemoglobin and oxyhemoglobin crystals to distinguish these compounds, but the photographs of crystals of carbonmonoxide hemoglobin and oxyhemoglobin of some species, such as guinea pig, show no appreciable difference. 3. Differences between the carbonmonoxide hemoglobins, as well as between the oxyhemoglobins, of the different species studied are indicated. 4. The carbonmonoxide hemoglobin crystals from the bloods studied are species specific in their nature, and, in many cases, can be distinguished from the analogous oxyhemoglobin by crystallographic study.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes

In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.     

T-cell-activating monoclonal antibodies, reacting with both leukocytes and erythrocytes, recognize the guinea pig Thy-1 differentiation antigen: characterization and cloning of guinea pig CD90

A glycophosphatidylinositol (GPI)-linked differentiation antigen expressed on guinea pig T and B lymphocytes was identified by several monoclonal antibodies; it has been shown previously that this membrane protein induced strong polyclonal T cell proliferation upon antibody binding and costimulation by PMA. Purification by immunoadsorption and microsequencing revealed that this T-cell-activating protein is the homologue of Thy-1 or CD90. In contrast to the Thy-1 antigen of most other species, guinea pig Thy-1 has a much higher molecular weight, which is due to a more extensive N-linked glycosylation, bringing the molecular weight of the total antigen up to 36 kDa. Molecular cloning of guinea pig Thy-1 indicated that the deduced molecular weight of the protein backbone is 12,777 after removal of an N-terminal 19-amino-acid leader peptide and cleavage of the 31 amino acids for GPI anchoring the C-terminal end. Sequence comparison showed that guinea pig Thy-1 has an 82% homology to human and a 72% homology to mouse Thy-1 on the amino acid level. Immunohistological staining of cryostat sections revealed intensive staining with the monoclonal antibody H154 on fibroblasts, fibrocytes, Kupffer cells, alveolar macrophages, and mesangial cells. As observed in the human, mouse, and rat, Thy-1 is abundant in the guinea pig brain. Unlike Thy-1 expression in other species, guinea pig Thy-1 is strongly expressed on most resting, nonactivated B cells and, to a lesser extent, on erythrocytes. While treatment of erythrocytes and lymphocytes with GPI-specific phospholipase C largely decreased reactivity with mAb H154, T cells retained the proliferative response to antibody and phorbol esters.     

Interaction of human Waldenstr?m's IgM proteins with guinea pig and human complement.

The activity of purified human Waldenstr?m's IgM protein to fix complement of human and guinea pig origins was compared at different temperatures using the polystyrene latex particle-adsorption method. It was shown that the interaction of the IgM proteins with complement differed depending on the source of complement and that a pronounced heterogeneity in complement-fixing activity was observed among the IgM proteins when tested with guinea pig complement. Thus, by the use of guinea pig complement, six human IgM proteins examined were classified roughly into two groups, one having a high and the other a low activity at 3 C as well as at 37 degrees C. With human complement, five proteins showed a rather uniform activity at 37 degrees C. However, there was one protein with no detectable activity, suggesting the presence of non-complement-fixing protein in the IgM class. All the six proteins showed no significant activity with human complement at 3 C. No antigenic difference has been found as yet in the Fc or Cmu2 region among these IgM proteins examined.