Growth, Cell Division, and Fragmentation in a Species of Flexibacter

Flexibacter FS-1, a gram-negative gliding bacterium was grown in liquid culture as long (over 100-mum) filaments. The filaments possessed a triple-track wall which resembled that found in other gram-negative bacteria. Although phase-contrast microscopy indicated that the long filaments were nonseptate, electron microscopy revealed three or four septa along the length of each filament. The septa contained lysozyme-sensitive, electron-opaque material, presumed to be peptidoglycan, sandwiched between cell membranes. The outer triple track wall was not part of the septum. Mesosomes were seen in various areas of the cell and frequently were observed attached to septa in different stages of completion. Studies of the organism in slide culture revealed that individual filaments grew in an exponential fashion and divided in the middle despite the long length and multiseptate condition. When the temperature of a liquid culture growing exponentially with a generation time of 90 minutes was shifted from 30 to 35 C, the filaments fragmented into three or four shorter cells within 120 min. The short cells continued to grow exponentially at 35 C at approximately the same rate as at 30 C. When the culture was shifted back to 30 C, the cells immediately stopped dividing and began to elongate. After a period of 2 to 3 hr, cell division resumed. It is suggested that the shift-up in temperature induced the completion of the cross wall (centripetal growth of the triple-track wall) and cell separation at the sites of previously formed septa, whereas the shift-down in temperature caused a transient inhibition of cross-wall formation but not of growth. Fragmentation was inhibited by sodium azide but took place despite the inhibition of protein synthesis by chloramphenicol or the inhibition of deoxyribonucleic acid synthesis by mitomycin C.     

TRH-like immunoreactivity in endocrine cells and neurons in the gastro-intestinal tract of the rat and guinea pig

Summary By use of the indirect immunofluorescence technique, the cellular localization of thyrotropin-releasing hormone (TRH) was studied in the gastrointestinal tract of rats and guinea pigs of different ages. TRH-like immunoreactivity (LI) was observed in many pancreatic islet cells of young rats and guinea pigs but only in single cells of 6-month-old rats. In aged guinea pigs, a reduction in the number of TRH-positive cells was evident; however, numerous strongly fluorescent cells were still present. In the guinea pig, TRH-LI was in addition observed in gastrin cells in the stomach. TRH-positive nerve fibers occurred in the myenteric plexus of the oesophagus, stomach and intestine of the rat, and in the muscle layers of the guinea pig. These results suggest a functional role of TRH both as hormone and neuroactive compound in various portions and sites of the gastro-intestinal tract of the rat and guinea pig     

Species comparison of adenosine A1 receptors in isolated mammalian atrial and ventricular myocardium

Various species have been used as models to study the effects of adenosine (ADO) on atrial and ventricular myocardium, but few direct tissue comparisons between species have been made. This study further characterizes adenosine A(1) receptor binding, adenylate cyclase activity and direct and indirect A(1) receptor-mediated functional activity in atrial and ventricular tissue from Sprague-Dawley rats and Hartley guinea pigs. Rat right atria (RA) were found to be significantly more sensitive to cyclopentyladenosine (CPA), while guinea pig left atria (LA) were more sensitive to CPA. After the addition of isoproterenol (ISO), the reduction of CPA response in rat RA was significantly greater than in guinea pig; however, after ISO treatment, the guinea pig LA was more sensitive to CPA than the rat. Adenylate cyclase inhibition by CPA was significantly greater in atria and ventricles obtained from guinea pig than rat. In competition binding experiments, guinea pig RA had significantly more high affinity sites than rat, but the K(i)s were not significantly different. There were no significant differences between guinea pig LA and rat LA. Guinea pig ventricular tissue had fewer high affinity sites than rat without any differences in their K(i) values. In antagonist saturation experiments, the density and affinity of A(1) receptors in guinea pig cardiac membranes were significantly greater than in rat. Our results indicate definite species differences as well as tissue differences between rat and guinea pig. These differences must be considered when interpreting studies using rat and guinea pig tissue as models for cardiac function.     

Identification of B2 bradykinin binding sites in the guinea pig nasal turbinate

Specific high affinity BK binding sites in the nasal turbinate of the guinea pig have been demonstrated. Specific [3H]BK binding (10-330 pM) was saturable, and nonlinear least squares analysis indicated the presence of a high affinity binding site with a Kd value of 60 (50-78) pM and a Bmax value of 13.1 = 2.0 fmol/mg protein. In inhibition experiments, D-Phe7-BK (a B2 antagonist) inhibited [3H]BK binding with a Ki value of 23 nM, while des-Arg9[Leu8]-BK (a B1 antagonist) had no effect up to a concentration of 10 microM. These studies indicate the presence of B2 BK receptors in the guinea pig nasal turbinate.     

Sites in Myelin Basic Protein that React with Monoclonal Antibodies

The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1-14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of overlapping epitopes includes the central Phe91-Phe92 sequence. One epitope is contained entirely within sequence 90-99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114-121, specifically including Trp118, and reacts with a rat IgG MAb. A cross-reacting epitope probably includes residues 44-45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131-140 but is severely species-restricted. This region in guinea pig BP evoked a species-specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat-18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.     

Nasal glandular secretory response to cholinergic stimulation in humans and guinea pigs.

A guinea pig model of nasal secretory responses was developed to assess the contributions of vascular permeability and glandular secretion responsible for the production of cholinergically stimulated nasal secretions. The nasal secretory responses to provocation with saline, methacholine, and atropine on the ipsilateral (challenged) side and contralateral (reflex) side were analyzed by measurement of total protein (Lowry method), guinea pig albumin (enzyme-linked immunosorbent assay), 125I-labeled bovine serum albumin after intravenous injection, and alkaline phosphatase enzyme activity in nasal fluid. Alkaline phosphatase was found to be localized to submucosal glands by zymography. Topical methacholine challenge increased the secretion of total protein, alkaline phosphatase activity, and albumin on the ipsilateral challenged side, whereas the percentage of total protein represented by albumin was not increased. This response was totally prevented by atropine pretreatment. Serial provocation with methacholine resulted in progressively reduced amounts of both the total protein and alkaline phosphatase in secretions. The observation that repeated challenges produced progressively smaller responses was also examined employing human nasal provocation. Repeating methacholine (25 mg) challenges four times at 10-min intervals in six human volunteers revealed that the initial challenge produced the largest response as reflected in total protein, albumin, lysozyme, lactoferrin, immunoglobulin (Ig) G, IgA, and secretory IgA secretion. When the constituents in secretions were analyzed in relationship to the total protein, the two vascular proteins, IgG and albumin, demonstrated the greatest decrements with repeated methacholine challenges. The glandular proteins, lactoferrin, lysozyme, and secretory IgA, either remained constant or increased in their relative proportion to total protein. Thus, cholinergic stimulation causes glandular secretion from both the guinea pig and human nasal mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the Binding of [3H]Ro 5-4864, a Convulsant Benzodiazepine, to Guinea Pig Brain

The density of high affinity binding sites for [3H]4'-chlorodiazepam [( 3H]Ro 5-4864) in guinea pig cerebral cortex is significantly higher (3.8-fold) than the density reported in the rat, and is nearly equal to the density of binding sites for other [3H]benzodiazepines (e.g., diazepam, flunitrazepam). The density of these [3H]Ro 5-4864 binding sites was generally higher in guinea pig brain than in rat brain, with the exception of olfactory bulb. Both the subcellular distribution and pharmacologic profile of these sites in guinea pig brain appears qualitatively similar to observations previously reported in the rat. The high density of binding sites for [3H]Ro 5-4864, coupled with the potency of this compound as a convulsant in the guinea pig, suggest this species will be a valuable model for elucidating putative pharmacologic and physiologic functions of these sites in brain.     

Grouping of 20 reference strains of Legionella species by the growth ability within mouse and guinea pig macrophages

20 Reference strains of Legionella species, isolated from human, were classified according to their ability to grow within thioglycolate-induced peritoneal macrophages of mice and guinea pigs. Inbred and congenic mice were used to study the effect of the natural resistance genes Lgn1 and Bcg that are expressed phenotypically in the mouse macrophages. The Lgn1 gene controlled the intracellular growth of Legionella pneumophila Philadelphia-1 and Legionella jordanis GIFU 12657, but the Bcg gene did not affect the intracellular growth of any organism examined. Based on these results and the growth ability in guinea pig macrophages, the 20 reference strains were divided into four groups. This grouping will help us to understand a variety of modes of interaction between Legionella species and macrophages.     

Growth of Myxovirus parainfluenza type 3 in organ cultures of guinea pig tissue

Craighead, J. E. (Harvard Medical School, Boston, Mass.). Growth of Myxovirus parainfluenza type 3 in organ cultures of guinea pig tissue. J. Bacteriol. 92:751-761. 1966.-Organ cultures of adult guinea pig nasal mucosa, lung, and pleura were infected with Myxovirus parainfluenza type 3. Observations were made on the growth of virus at intervals after inoculation. An inoculum of 10(2.5) tissue culture infectious doses (tcid(50)) initiated infection in each of the tissues. Cultures of nasal mucosa yielded up to 10(6.0)tcid(50) per 6 hr for periods of as long as 2 weeks. Virus production was not affected by the "immune" status of the animal used as a source of tissue. Introduction of antiserum into the medium appeared to suppress virus release but failed to "cure" the infection. Interferon was not detected in fluids bathing the nasal mucosa. Cultured fragments of lung produced virus for 28 days after inoculation. As much as 10(5.0)tcid(50) per 6 hr was released by the tissue. Pleural mesothelial cells lining the diaphragm yielded up to 10(6.0)tcid(50) per 6 hr over a 14-day period. Histological sections showed that the tissues retained differentiated morphological features during maintenance in vitro. Cytological changes unequivocally associated with infection were not recognized. The techniques described give reproducible, quantitative results. Organ cultures are feasible for the study of virus growth and cytopathology in differentiated tissues.     

Decidual cell reaction: relative response to traumatization in uterus of rat, hamster and guinea pig

Relative decidual cell response to traumatization was studied in the uterus of immature rat, hamster and guinea pig. A single dose of progesterone (1.0 mg/animal) caused 285 +/- 41 and 459 +/- 58% uterotrophy in traumatized horn of rat and hamster respectively, while in guinea pig increasing doses of progesterone (1, 2 and 5 mg/animal), several combinations of estrogen and progesterone or alteration in treatment schedule of the two hormones as well as extending the day of traumatization failed to elicit comparable uterotrophic response. Uterus of immature hamster was found to be most responsive to traumatization and that of guinea pig the least.     

Molecular Cloning, Expression, and In Silico Structural Analysis of Guinea Pig IL-17

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.     

Cervical lymphadenitis in guinea pigs: infection via intact ocular and nasal mucosa by Streptococcus zooepidemicus.

The traditional view regarding the pathogenesis of cervical lymphadenitis in guinea pigs is that Lancefield Group C Streptococcus gains access to cervical lymph nodes via an abraded oral mucosa. In this study, it is established that inoculation of intact nasal and conjunctival mucous membranes with Streptococcus zooepidemicus (Lancefield Group C) also can produce the disease. Weanling (SPF) guinea pigs (Cavia porcellus) were divided into two experimental groups of 10 and two control groups of four each. Guinea pigs from each group were individually housed in separate cubicles. Group I was inoculated with 0.05 ml of culture containing 2.8 x 10(7) CFU/ml of S. zooepidemicus into the conjunctiva of the left eye. Group II received a similar inoculum into the left nares. Control groups received 0.05 ml of TSB broth in the same sites. Five of ten guinea pigs in Group II died four to nine days postinoculation. Surviving guinea pigs were euthanatized at intervals between days 4-13 postinoculation. All guinea pigs were necropsied, cultured and examined for evidence of infection. S. zooepidemicus was recovered from 30/50 and 39/46 sites cultured from Groups I and II, respectively. Lymphadenitis was found in cervical lymph nodes from 8/10 guinea pigs in Group I and 10/10 in Group II. The conjunctival and nasal mucosa, therefore, represent potential sites of entry resulting in cervical lymphadenitis in guinea pigs.     

Species differences in hepatic peroxisome proliferation, cell replication and transforming growth factor-beta1 gene expression in the rat, Syrian hamster and guinea pig

The objective of this study was to evaluate species differences in the hepatic effects of three potent rodent peroxisome proliferators, namely methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY), particularly with respect to effects on replicative DNA synthesis and transforming growth factor-beta1 (TGF-beta1) gene expression. Male Sprague-Dawley rats, Syrian hamsters and Dunkin-Hartley guinea pigs were given daily oral doses of 0 (corn oil) and 75 mg/kg MCP for periods of 6 and 21 days. Syrian hamsters and guinea pigs were also treated with 25 mg/kg CIP and 25 mg/kg WY. Relative liver weights were significantly increased in peroxisome proliferator-treated rats and Syrian hamsters, but not in guinea pigs. Hepatic peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities and CYP4A isoform mRNA levels were significantly increased in rats and Syrian hamsters, whereas only minor effects were observed in the guinea pig. Replicative DNA synthesis was studied by implanting 7-day osmotic pumps containing 5-bromo-2'-deoxyuridine during study days -1 to 6 and 14 to 21. Hepatocyte labelling index values were increased by MCP in the rat, but neither MCP, CIP nor WY produced any significant effect on replicative DNA synthesis in the Syrian hamster and guinea pig. MCP treatment increased TGF-beta1 and insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor gene expression in the rat. In the Syrian hamster, effects on TGF-beta1 and IGFII/Man6P receptor gene expression were also observed in some instances, whereas TGF-beta1 mRNA levels were essentially unchanged in the guinea pig. These results provide further evidence for marked species differences in response to rodent peroxisome proliferators. While peroxisome proliferators produce a wide spectrum of effects in rat liver, other species such as the Syrian hamster and guinea pig are less responsive and in the case of some endpoints (e.g., cell replication) may be refractory.     

Comparison of rotavirus strains by hemagglutination inhibition.

Rotaviruses have been shown to be of importance as aetiologic agents of gastroenteritis in infants and in domestic animals of several species. Hemagglutinins were prepared from two Canadian isolates of bovine rotavirus and from one isolate of a simian rotavirus. A United Kingdon isolate of bovine rotavirus was shown not to possess hemagglutinating activity, indicating a strain difference between a Canadian and United Kingdom bovine rotavirus. In hemagglutination-inhibition (HAI) tests a rabbit hyperimmune (two injections) serum, prepared to one of the bovine rotaviruses, was not helpful in distinguishing the two bovine viruses because of cross-reactions between the viruses. However, it was possible to distinguish the bovine viruses from the simian virus with this serum. When guinea pig immune sera were prepared to the four rotavirus strains and tested with the three hemagglutinins in the HAI test, antigenic differences between the four strains of rotavirus were demonstrated. Hyperimmune guinea pig serum prepared to a strain of human rotavirus did not inhibit any of three hemagglutinins indicating that the human strain is different from the three rotavirus strains which gave hemagglutinins.     

Cytotoxic effects of normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation.

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca²⁺, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca²⁺ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg²⁺ These observations also highlight the Mg²⁺-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

The identification, characterization, and distribution of guinea pig CCR4 and epitope mapping of a blocking antibody.

Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon gamma. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on approximately 12% of guinea pig peripheral blood T helper cells, and CCR4(+) guinea pig thymocytes were detected in low numbers. However, CCR4(+) T helper cells constituted approximately 9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.