Antigen-specific T helper clones in a nonresponder strain require augmentation for expression of helper activity. Evidence for a possible antigen presentation defect in B cells

Hen egg-white lysozyme (HEL)-specific Thy-1+, Lyt-1+2- T cell lines and clones were derived from the nonresponder C57BL/6 strain. Although the antigen-specific proliferative response of these T cells in the presence of syngeneic irradiated spleen cells as a source of antigen-presenting cells (APC) was normal, the same cells were incapable of stimulating B cells to secrete antibody in vitro. This deficiency could, however, be corrected by the addition of an excess of normal T cells or a supernatant from concanavalin A-stimulated rat spleen cells. Alternatively, the use of highly cross-reactive ring-necked pheasant lysozyme in the cultures allowed expression of efficient help, ruling out any inherent deficiency in the T cells. The antibody response was specific and required MHC compatibility between the T lines and responding B cells. By using (H-2b X H-2d)F1 B cells and another H-2d-restricted HEL-specific T line, it was shown that only the H-2b-restricted T-B collaboration required exogenous factors, and the H-2d-restricted collaboration did not. Because both proliferative and helper responses are dependent upon MHC-restricted antigen presentation by macrophage-APC and B cells, respectively, these results suggest that the defect in the nonresponder H-2b-restricted T-B collaborative pathway may relate to the inability of B cells to adequately process and present HEL to clonal T cells.     

Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

MMP-9 gene ablation mitigates hyperhomocystenemia-induced cognition and hearing dysfunction

Hyperhomocysteinemia (HHcy) is associated with cognitive decline and hearing loss due to vascular dysfunction. Although we have shown that HHcy-induced increased expression of matrix metalloproteinase-9 (MMP-9) is associated with cochlear pathology in cystathionine-β-synthase heterozygous (CBS^+/?) mice, it is still unclear whether MMP-9 contributes to functional deficit in cognition and hearing. Therefore, we hypothesize that HHcy-induced MMP-9 activation causes vascular, cerebral and cochlear remodeling resulting in diminished cognition and hearing. Wildtype (WT), CBS^+/?, MMP-9^?/? and CBS^+/?/MMP-9^?/? double knock-out (DKO) mice were genotyped and used. Doppler flowmetry of internal carotid artery (ICA) was performed for peak systolic velocity [PSV], pulsatility index [PI] and resistive index [RI]. Cognitive functions were assessed by Novel Object Recognition Test (NORT) and for cochlear function Auditory brainstem response (ABR) was elicited. Peak systolic velocity, pulsatility and resistive indices of ICA were decreased in CBS^+/? mice, indicating reduced perfusion. ABR threshold was increased and maximum ABR amplitude and NORT indices (recognition, discrimination) were decreased in CBS^+/? mice compared to WT and MMP-9^?/?. All these parameters were attenuated in DKO mice suggesting a significant role of MMP-9 in HHcy-induced vascular, neural and cochlear pathophysiology. Regression analysis of PSV with ABR and cognitive parameters revealed significant correlation (0.44–0.58). For the first time, MMP-9 has been correlated directly to functional deficits of brain and cochlea, and found to have a significant role. Our data suggests a dual pathology of HHcy occurring due to a decrease in blood supply (vasculo-neural and vasculo-cochlear) and direct tissue remodeling.     

Spatial and temporal organization of actin during hydration,activation, and germination of pollen inPyrus communis L.: a population study

Summary Dynamics of F-actin organization during activation and germination ofPyrus communis (pear) pollen was examined using rhodaminephalloidin. Prior to activation, the rhodamine-phalloidin labelling pattern appeared as circular profiles in the peripheral cytoplasm of the vegetative cell and as coarse granules around the vegetative nucleus. In activated pollen, parallel arrays of cortical F-actin were aligned circumferentially, along the polar axis in non-apertural areas of the pollen grain, and at 45° to 90° to the polar axis beneath the apertures. Some pollen also showed fluorescent granules or fusiform bodies dispersed throughout the cytoplasm, but as the number of such pollen diminished with prolonged incubation, these are being considered as intermediate patterns. In later stages, the filaments became organized as interapertural bundles traversing the three apertures. However, prior to emergence of the pollen tube, labelling became confined to a single aperture. In germinated pollen grains, actin microfilaments are aligned more or less axially with respect to the axis of the developing pollen tube.The granular labelling pattern seen around the vegetative nucleus prior to pollen activation also became clearly filamentous with pollen activation; this filamentous pattern persisted until germination when it was replaced by cables that aligned longitudinally with respect to the emerging tube axis.The results demonstrate that the organization of actin undergoes considerable changes in the period preceding pollen germination and that microfilament polarization is achieved before pollen germination.     

Esterase-6 polymorphism inDrosophila melanogaster:Effects of temperature and methyl malonate on genotypic trajectories in polymorphic populations set up with highly inbred lines

It is generally difficult to identify possible effects of selection at a specific locus because of the heterogeneity of the genetic background. Geographical patterns ofEst-6 gene frequencies suggest that there is selection at this locus but selection on loci closely linked to it cannot be excluded. Differences in catalytic properties between allozymes have been shownin vitro; further, several laboratory studies have shown apparent fitness differences between allozymes. Our study used inbred lines highly homogeneous in the genetic background. Four populations were set up fromEst-6s andEst-6F homozygous females inseminated by males of the same genotype at each combination of three factors: temperature (18 and 25°C); methyl malonate (presence or absence); input gene frequencies [p(S) = 0.2 and 0.8]. The populations were sampled periodically for about 28 generations. Methyl malonate was chosen to exert pressure in the enzymatic function of esterase-6. Statistical analyses show that: there are no sex differences; gene frequencies change from input values to those of the first sampling, when only individuals of the first generation are present at 18^oC or individuals of the second generation just begin to appear at 25°C; gene frequencies do not change thereafter and Hardy-Weinberg equilibrium is established. The changes in gene frequencies observed in the first generations suggest thatEst-6 can under certain conditions be a target of selection. Such conditions may not, however, occur in natural populations.     

The choice between two distinct T cell determinants within a 23-amino acid region of lysozyme depends on their structural context

The specificity of C57BL/6 T cells reactive to peptide aa 74-96 of hen egg-white lysozyme (HEL) was analyzed by using a panel of synthetic peptides of varying lengths from this region. It was found that peptide 74-96-reactive T cells induced by native HEL (aa 1-129) or its denatured fragment L2 (aa 13-105) recognized two distinct but overlapping determinants contained within aa 74-90 or aa 81-96, respectively. Peptide 74-96 itself induced both peptide 74-90-and peptide 81-96-specific T cells. Thus, a choice was made between these two potential T cell determinants on peptide 74-96, depending on which immunogen was used. Interestingly, the ability of both peptide determinants aa 74-90 and aa 81-96 to stimulate peptide 74-96-reactive T cells was partly dependent on the presence of residues within the overlap region (aa 81-90), suggesting that this region may play an important role in Iab-restricted T cell activation. This was further supported by the poor immunogenicity of shorter peptides 74-86 or 85-96, lacking residues from the overlap region in B6 mice. These two short peptides were nevertheless capable of eliciting T cell responses in B10.A mice, suggesting that the importance of this overlap region in obtaining a response to peptide 74-96 is related to the MHC haplotype.     

A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice

E thiraj , S. & S uresh , E.R. 1985. A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice. Journal of Applied -Bacteriology 59 , 239–242.
A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos . This is the first report of L. oenos as a spoilage organism in fruit products other than wine.     

A limited region within hen egg-white lysozyme serves as the focus for a diversity of T cell clones

The C57BL/6 (H-2b) mouse is a nonresponder to hen egg-white lysozyme (HEL) injected i.p., owing to a T suppressor cell-inducing determinant at the amino-terminal region. After immunization with a 93-amino acid fragment (a.a. 13-105) of HEL lacking this determinant, all clones from two independently derived C57BL/6 T cell lines were found to be specific for epitopes within a subregion of peptide 74-96. Three specificity patterns for the clones could be defined on the basis of cross-reactivities with only two other species variant lysozymes. Reactivities of all three specificity groups was consistent with the serine to threonine substitution at position 91, although reactivity of one of the groups could be affected by substitutions at position 84. The results confirm at the clonal level that even for distantly related antigens, only limited regions are recognized by T cells. They are consistent with the notion that specific sites on the antigen capable of interaction with Ia molecules lead to dominance of certain regions for T cell reactivity. Moreover, the diversity in specificity among clones suggests that the limiting feature of T cell responsiveness is not a lack of available T cells in the repertoire directed against a single antigenic site.     

Control of ethanol production by yeast: Role of pyruvate decarboxylase and alcohol dehydrogenase

Summary The critical role of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in determining the rate of ethanol production is confirmed using PDC constitutive mutants. By deriving strains with altered levels of these two enzymes, it has been found that a high level of both PDC and ADH is necessary for faster ethanol production.