Infant milk feeding and bone health in later life: findings from the Hertfordshire cohort study.

Using data from the Hertfordshire cohort study, this study examined the effect of breastfeeding and bottle feeding on adult lumbar spine and femoral neck bone mineral content (BMC) and bone mineral density (BMD). The type of infant milk feeding was significantly associated with lumbar spine BMD in males. INTRODUCTION: Using data from the Hertfordshire cohort study (HCS), this study aims to examine the effect of infant milk feeding on bone health in later life by comparing the effect of breastfeeding and bottle feeding on lumbar spine and femoral neck BMC and BMD. METHODS: Information about infant milk feeding, birth weight (kg) and weight at 1 (kg) was collected by health visitors between 1931 and 1939 in Hertfordshire. BMC and BMD measurements were taken by DXA scan between 1998 and 2004. Linear regression models adjusted for conditional weight at 1, age at DXA scan, sex, adult BMI, smoking behaviour, alcohol consumption, physical activity, dietary calcium, and prudent diet score. RESULTS: Infant milk feeding was significantly associated with lumbar spine BMD (b = - 0.028; 95% CI, - 0.055; - 0.000; p value, 0.047) in males. On average, males who consumed breastmilk alternatives in infancy had lower lumbar spine BMD measurements than those who were fed only breastmilk. These associations remained significant in fully adjusted models. There were no significant associations between infant milk feeding and bone health for females. CONCLUSIONS: Significant associations between infant milk feeding and lumbar spine BMD in males indicate that breastmilk may be protective for the bone health of male babies. The evidence presented here underscores the potential lifelong benefits of breastfeeding and may highlight the differences between osteoporotic risk factors for males and females.

Four pediatric patients with autosomal recessive polycystic kidney disease developed new-onset diabetes after renal transplantation.

NODAT is increasingly prevalent. Compared with adult recipients, NODAT is less prevalent in pediatric renal transplant recipients; however, some risk factors for its development in young patients have been defined. We report four pediatric renal transplant recipients with ARPKD who developed NODAT. We review the current pediatric NODAT literature and hypothesize that ARPKD may be an additional risk factor for NODAT.

Lipoprotein lipase is frequently overexpressed or translocated in cervical squamous cell carcinoma and promotes invasiveness through the non-catalytic C terminus.

BACKGROUND: We studied the biological significance of genes involved in a novel t(8;12)(p21.3;p13.31) reciprocal translocation identified in cervical squamous cell carcinoma (SCC) cells. METHODS: The rearranged genes were identified by breakpoint mapping, long-range PCR and sequencing. We investigated gene expression in vivo using reverse-transcription PCR and tissue microarrays, and studied the phenotypic consequences of forced gene overexpression. RESULTS: The rearrangement involved lipoprotein lipase (LPL) and peroxisome biogenesis factor-5 (PEX5). Whereas LPL-PEX5 was expressed at low levels and contained a premature stop codon, PEX5-LPL was highly expressed and encoded a full-length chimeric protein (including the majority of the LPL coding region). Consistent with these findings, PEX5 was constitutively expressed in normal cervical squamous cells, whereas LPL expression was negligible. The LPL gene was rearranged in 1 out of 151 cervical SCCs, whereas wild-type LPL overexpression was common, being detected in 10 out of 28 tissue samples and 4 out of 10 cell lines. Forced overexpression of wild-type LPL and PEX5-LPL fusion transcripts resulted in increased invasiveness in cervical SCC cells, attributable to the C-terminal non-catalytic domain of LPL, which was retained in the fusion transcripts. CONCLUSION: This is the first demonstration of an expressed fusion gene in cervical SCC. Overexpressed wild-type or translocated LPL is a candidate for targeted therapy.

The effect of broken conjugation on the excited state: ether linkage in the cyano-substituted poly(p-phenylene vinylene) conjugated polymer poly(2,5,2',5'-tetrahexyloxy-8,7'-dicyano-di-p-phenylene vinylene).

We investigate the effect of broken conjugation on the excited state dynamics of excimers in cyano-substituted phenylene-vinylene polymers. We compare previous studies on the well-characterized poly(2,5,2',5'-tetrahexyloxy-8,7'-dicyano-di-p-phenylene vinylene) (CN-PPV) with poly[oxa-1,4-phenylene-1,2-(1-cyano)-ethenylene-2,5-dioctyloxy-1,4-phenylene-1,2-(2-cyano)-ethenylene-1,4-phenylene] (CN-ether-PPV), in which the conjugation is disrupted by the insertion of an oxygen atom within the polymer backbone. Despite the broken conjugation, the spectroscopic behavior of the two materials is similar, indicating that the cyano group dominates the photophysics in these materials. The emission in CN-ether-PPV is due to a single-chain exciton in solution and due to an interchain excimer in thin film, as previously reported for CN-PPV; however, the excimer absorption and emission in thin film are blueshifted by approximately 0.2 eV relative to CN-PPV, implying that the excimer in CN-ether-PPV is less stable. Furthermore, substitution of an ether group along the chain results in decay times in both solution and film that are twice as long than in CN-PPV due to the broken conjugation which restricts the exciton within a conjugation segment and reduces its access to internal quenching sites. These properties result in a decay time of 14 ns for CN-ether-PPV film, one of the longest decay times observed in a conjugated polymer film. The long lifetime indicates a large exciton diffusion length, making these species particularly vulnerable to quenching by other materials. This work has implications for the design of conjugated polymers for efficient optoelectronic devices, such as photovoltaics.

The relationship of the transcutaneous oxygen tension, pulse waves and systolic pressures to the risk for limb amputation in patients with peripheral arterial disease and skin ulcers or gangrene.

AIM: Our aim was to determine how the risk associated with presence of low transcutaneous oxygen tension (tcPO2) for subsequent major amputation in patients with skin ulcers or gangrene and peripheral arterial disease, compares with the risks associated with low peripheral pressures and low amplitude of pulse waves. Secondly, we determined whether combination of measurements of oxygen tension with that of the pressures or pulse wave amplitude predicts amputation better than pressure, wave or oxygen tension measurements alone. METHODS: Measurements were carried out to obtain foot tcPO2, ankle and toe pressures, pressure indices, and toe pulse wave amplitude in 75 limbs with skin lesions and arterial disease of 66 patients referred to the vascular laboratory. These variables were related to the risk of a subsequent major amputation during a median time of 4.2 years, using Cox proportional hazards model. RESULTS: Low oxygen tension was associated with increased risk of amputation (relative risks 2.16 and 2.55 for tcPO2 < or = 10 mmHg and < or = 20 mmHg, respectively, P<0.05; relative risk 2.22 for tcPO2 < or = 30 mmHg, P=0.07). The relative risks associated with cutoff values of ankle and toe pressures and pressure indices varied from 2.53 (toe < or = 20 mmHg, P<0.05) to 5.83 (ankle < or = 50 mmHg, P<0.001) and the relative risk associated with low wave amplitude (< or = 4 mm) was 3.41, P<0.01. The cutoff values of tcPO2 became insignificant when included in the models together with each pressure variable or pulse amplitude separately. In contrast, wave amplitude remained significantly associated with increased risk of amputation after controlling for each pressure variable (P<0.05). CONCLUSIONS: TcPO2 < or = 10 mmHg and < or = 20 mmHg are related significantly to increased risk of amputation in patients with skin lesions and arterial disease, but these relative risks are similar in magnitude or smaller than those associated with low cutoff values of pressures, pressure indices or pulse wave amplitude. Low wave amplitude does provide significant information in addition to peripheral pressures with respect to the risk of amputation. On the other hand, low tcPO2 does not provide significant information in addition to peripheral pressures or pulse wave amplitude.

Electrochemical and optical characterization of p- and n-doped poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene].

We study electrochemical p- and n-type doping in the well-known light-emitting polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV). Doping reactions are characterized using cyclic voltammetry. Optical measurements including photoluminescence and UV/Vis/NIR transmission were performed on doped samples. We find that oxidation in MEH-PPV is a highly reversible reaction resulting in stable freestanding doped films, while the reduced form is unstable and the reaction irreversible. We discuss the dependence of doping reactions on scan rate, film thickness, salt type and concentration, and working electrode type. We observe the development of two additional broad absorption bands in both lightly and heavily doped films accompanied by a slight blueshift in the primary optical transition, suggesting bipolaron band formation. Finally we find that both p and n dopings result in extremely sensitive photoluminescence quenching. We propose a physical model for understanding electrochemical doping in MEH-PPV and the implications this has on the development of such technologies as polymer light-emitting electrochemical cells, electrochromic devices, actuators, and sensors.

Infections of the subcutis and skin of dogs caused by rapidly growing mycobacteria.

Nine dogs with panniculitis due to rapidly growing mycobacteria (RGM) were examined over 17 years. Dogs were two to 15 years; five were male, four were female. All were obese or in good condition. Antecedent injury, typically a dog bite or vehicular trauma, could be identified in some patients, while one bitch had hyperadrenocorticism. Infections involved different locations, although the cervicothoracic region, dorsum or flank were most often affected. Patients were systemically well, apart from one dog with pyrexia and two with pain or lameness. Cytology demonstrated pyogranulomatous inflammation, but in only one case was it possible to see acid-fast bacilli (AFB) in smears. Histology demonstrated chronic active pyogranulomatous panniculitis and dermatitis; AFB could be detected in only four specimens. Culture of aspirates or resected tissues demonstrated RGM in all cases, comprising six Mycobacterium smegmatis group and three Mycobacterium fortuitum group isolates. Resection of infected tissues, perioperative injectable antimicrobials and long courses of oral antimicrobials chosen according to susceptibility data generally effected a cure, although some cases recurred.

The value of toe pulse waves in determination of risks for limb amputation and death in patients with peripheral arterial disease and skin ulcers or gangrene.

OBJECTIVES: The purpose of this study was to determine whether the presence of low amplitude of pulse waves recorded from the toes is related to the risk of subsequent amputation and death in patients with skin ulcers or gangrene and peripheral arterial disease, and how the risk of low wave amplitude relates to the risk associated with low peripheral pressures. METHODS: A total of 309 patients with 346 limbs with skin lesions and arterial disease referred to the vascular laboratory were followed up for an average of 5 years (range, 1-8 years). Measurements were carried out to obtain ankle and toe pressures, pressure indices, and toe pulse wave amplitude. These variables were related to the risks of major amputation and total and cardiovascular death by means of the Cox proportional hazards model. RESULTS: Low toe pulse wave amplitude (< or = 4 mm) was associated with increased risk of amputation (relative risks 4.20 in all limbs and 2.63 in those with toe pressure < or = 30 mm Hg; P <.01). Wave amplitude remained significantly associated with increased risk of amputation after controlling for each pressure variable (P <.01). Low pulse wave amplitude and toe/brachial index were associated with increased risks of both total and cardiovascular death in all patients (relative risks ranged from 1.43-1.73; P <.05) and in those with toe pressure of 30 mm Hg or less (relative risks 1.56-1.90; P <.05). CONCLUSIONS: Low toe pulse wave amplitude is related significantly to increased risks of amputation and death in patients with skin lesions and arterial disease. The presence of low wave amplitude provides significant information in addition to peripheral pressures with respect to the risk of amputation.

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana.

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.

Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean.

Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily that is expressed predominantly in skeletal muscle. However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism. However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.

Skin perfusion pressure of the foot is a good substitute for toe pressure in the assessment of limb ischemia.

PURPOSE: Noninvasive measurements of limb systolic pressures are used routinely in the assessment of the severity of peripheral arterial disease, including the evaluation for critical limb ischemia. However, ankle pressures cannot be measured reliably in patients with medial calcification, which is especially common among patients with diabetes. Skin lesions on the toes or previous digital amputations may preclude the measurement of toe pressures. Measurements of skin perfusion pressure (SPP) are not subject to such limitations and were shown to be useful in the assessment of the severity of peripheral arterial disease. Because toe pressure is often used in the evaluation of severity of arterial disease and in the assessment for critical ischemia, we undertook to study whether there is a sufficient correlation between toe pressure and foot SPP that would allow the use of SPP measurements when toe pressures cannot be measured. METHODS: Measurements were carried out in 85 limbs of 71 patients referred to the vascular laboratory for evaluation for peripheral arterial disease. Diabetes mellitus was present in 43 patients. Each patient had foot SPP and toe pressure measurements. Toe pressures measured with photoplethysmography were correlated with foot SPP measured with laser Doppler scanning. RESULTS: There was a strong linear correlation between SPP and toe pressure (r = 0.87; P <.01). Also, significant correlation was found in both the patients with diabetes and the patients without diabetes (r = 0.85 and 0.93, respectively; P <.01 in both cases). CONCLUSIONS: We concluded that SPP measured in the foot correlates well with toe pressure and can be substituted for toe pressure measurement in patients in whom toe pressures cannot be measured.

Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy.

Aggregation of Ig light chains to form amyloid fibrils is a characteristic feature of light-chain amyloidosis, a light-chain deposition disease. A recombinant variable domain of the light chain SMA was used to form amyloid fibrils in vitro. Fibril formation was monitored by atomic force microscopy imaging. Single filaments 2.4 nm in diameter were predominant at early times; protofibrils 4.0 nm in diameter were predominant at intermediate times; type I and type II fibrils 8.0 nm and 6.0 nm in diameter, respectively, were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments, respectively. These observations support a model in which two filaments combine to form a protofibril, two protofibrils intertwine to form a type I fibril, and three filaments form a type II fibril.

ATP2A2 mutations in Darier's disease: variant cutaneous phenotypes are associated with missense mutations, but neuropsychiatric features are independent of mutation class.

Darier's disease (DD) is an autosomal dominant skin disorder characterized clinically by multiple keratotic papules, and histologically by focal loss of adhesion between epidermal cells (acantholysis) and by abnormal keratinization. Variant forms of cutaneous phenotype, sometimes familial, have been described. Associated neuropsychiatric features, including mental handicap, schizophrenia, bipolar disorder and epilepsy, have also been reported. The cause of DD was shown recently to be mutation in the ATP2A2 gene at 12q24.1, which encodes the sarco-endoplasmic reticulum calcium ATPase type 2 (SERCA2). Here, we show that while both common isoforms of SERCA2 are expressed in the cytoplasm of cultured keratinocytes and fibroblasts, in adult skin sections only the longer isoform, SERCA2b, was expressed abundantly in epidermal structures. Extended mutation analysis in European DD patients using single-strand conformation polymorphism and/or direct sequencing identified 40 different patient-specific mutations in 47 families. The majority (23/40) were likely to result in nonsense-mediated RNA decay. The remaining 17 were missense mutations distributed throughout the protein and were associated significantly with atypical clinical features. The clearest association was with the familial haemorrhagic variant where all four families tested had a missense mutation. Three of the families (one Scottish family and two unrelated Italian families) exhibited the same N767S substitution in the M5 transmembrane domain, and a fourth family, from Sweden, had a C268F substitution in the M3 transmembrane domain. Neuropsychiatric features did not appear to be associated with a specific class of mutation and may be an intrinsic, but inconsistent, effect of defective ATP2A2 expression.

Refined genetic mapping of the darier locus to a <1-cM region of chromosome 12q24.1, and construction of a complete, high-resolution P1 artificial chromosome/bacterial artificial chromosome contig of the critical region.

Darier disease (DD) (MIM 124200) is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells and by abnormal keratinization. We present linkage analysis showing, in four families, key recombination events that refine the location of the DD locus on chromosome 12q23-24.1 to a region of <1 cM. We have constructed a YAC/P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC)-based physical map that encompasses this refined DD region. The map consists of 35 YAC, 69 PAC, 16 BAC, and 2 cosmid clones that were ordered by mapping 54 anonymous sequence-tagged sites. The critical region is estimated to be 2.4 Mb in size, with an average marker resolution of 37.5 kb. The refinement of the critical interval excludes the ALDH2, RPL6, PTPN11, and OAS genes, as well as seven expressed sequence tags (ESTs) previously mapped in the DD region. The three known genes (ATP2A2, PPP1CC, and SCA2) and the 10 ESTs mapped within the critical region are not obvious candidates for the DD gene. Therefore, this detailed integrated physical, genetic, and partial transcript map provides an important resource for the isolation of the DD gene and, possibly, other disease genes.

The challenge and importance of defining critical limb ischemia.

Patients with critical limb ischemia are at risk of limb loss and have very high cardiovascular and total mortality rates, which are greater than can be accounted for by the usual risk factors for atherosclerosis. A definition of critical ischemia is necessary to determine the natural history of the disease and to assess and compare the efficacy of various forms of therapy. The definition needs to be based on hemodynamic criteria because clinical manifestations and outcomes are not reliable. The reasons for the difficulties in arriving at a generally acceptable definition are explored. Building on the previous work of others, modified hemodynamic definitions for critical and subcritical ischemia, which include measurements of pressures and of indices of microcirculation, are proposed.

Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.

The 126- and 183-kilodalton proteins of tobacco mosaic virus, and not their common nucleotide sequence, control mosaic symptom formation in tobacco.

Nucleotide substitutions at two positions within the open reading frame encoding the 126-kDa protein in the attenuated masked (M) strain of tobacco mosaic tobamovirus (TMV) to those found in the virulent U1-TMV genome led to the induction of near U1-TMV-like symptoms on leaves of Nicotiana tabacum L. cv. Xanthi nn by progeny virus (M. H. Shintaku, S. A. Carter, Y. Bao, and R. S. Nelson, Virology 221:218-225, 1996). In this study, further site-directed mutations were made at these positions within the M strain cDNA to determine whether the protein or nucleotide sequence directly controlled the symptom phenotype. The protein and not the nucleotide sequence directly controlled the symptom phenotype when amino acid 360 within the 126-kDa protein sequence was altered and likely controlled the symptom phenotype when amino acid 601 was altered. The effects of the substitutions at amino acid position 360 on viral protein accumulation were studied by pulse-labeling proteins in infected protoplasts. Accumulation of the 126- and 183-kDa proteins was less for an attenuated mutant than for two virulent mutants, but the viral movement protein and coat protein accumulated to levels reported to be sufficient for normal systemic symptom development. The size of necrotic local lesions on N. tabacum L. cv. Xanthi NN was negatively correlated with symptom development and accumulation of the 126-kDa protein for these mutants. With reference to this last finding, an explanation of the cause of the differing symptoms induced by these viruses is presented.

Value of toe pulse waves in addition to systolic pressures in the assessment of the severity of peripheral arterial disease and critical limb ischemia.

PURPOSE: Although pressure measurements are useful in the assessment of the severity of the arterial obstruction, they do not completely identify limbs with and without critical limb ischemia. Our objective was to test whether addition of the measurements of toe pulse waves (PW), which depend on distal perfusion, to pressure measurements could improve the determination of the severity of arterial disease and the presence of critical limb ischemia. METHODS: We measured toe pressure (TSP) and ankle/brachial index (ABI) and recorded PW with photoplethysmography in 358 limbs of 182 patients. RESULTS: TSP, ABI, and PW amplitude were lower in 67 limbs with rest pain, skin lesions, or both, with mean differences of 29 mm Hg, 0.12, and 16 mm, respectively (p < 0.01). Similarly, in the subgroup of 107 limbs with TSP < or = 30 mm Hg, TSP, and PW amplitude, but not ABI, were lower in 53 limbs with rest pain, skin lesions, or both, with mean differences of 10 mm Hg and 7 mm (p < 0.01). Multiple logistic regression showed that after controlling was done for TSP and ABI, the odds ratio for the presence of rest pain, skin lesions, or both associated with PW amplitude < or = 4 mm was 4.3 (95% confidence interval 1.7, 11.0; p < 0.01). In the subgroup with TSP < or = 30 mm Hg, this odds ratio was 3.5 (95% confidence interval 1.0, 11.6; p < 0.05). CONCLUSIONS: The findings indicate that addition of PW recording to pressure measurements is likely to increase the accuracy of assessment for critical limb ischemia.

Mapping nucleotides in the 126-kDa protein gene that control the differential symptoms induced by two strains of tobacco mosaic virus.

The differential symptom determinants of the Holmes' masked (M) and U1 strains of tobacco mosaic virus previously were mapped to the 5'-coterminal open reading frame (ORF) encoding the 126-kDa protein and the N-terminal two-thirds of the 183-kDa protein. Both proteins influence viral RNA accumulation, but the function of, and impact on, symptom formation by large domains within the 126-kDa gene, which are not conserved with sequences in analogous ORFs from other related viruses, are unknown. In the current study, cDNA clones representing each strain (i.e., MIC-TMV and U1-TMV) were mutated in these nonconserved domains to further define the nucleotides responsible for mosaic symptom induction on Nicotiana tabacum. Progeny virus of a mutant containing only eight nucleotide substitutions from the MIC-TMV sequence to the U1-TMV sequence within the 126-kDa protein ORF of MIC-TMV induced U1-TMV-like symptoms. Single or multiple substitutions among these eight nucleotides further defined residues critical for symptom modulation. Complementary substitutions in the MIC-TMV and U1-TMV sequences did not always yield progeny virus that induced complementary visual symptoms. Progeny of some mutants contained second-site spontaneous mutations at specific positions shown to influence symptom phenotype. For a subset of the stable site-directed mutants, there was no correlation between severity of systemic symptoms and chlorotic lesion size or virus accumulation in these chlorotic lesions on inoculated leaves.